Synthesis and evaluation of new Hsp90 inhibitors based on a 1,4,5-trisubstituted 1,2,3-triazole scaffold

Article

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Synthesis and Evaluation of New Hsp90 Inhibitors Based

on a 1,4,5-Trisubstituted 1,2,3-Triazole Scaﬀold

Maurizio Taddei,*^,†Serena Ferrini,^†Luca Giannotti,^†Massimo Corsi,^†,∥Fabrizio Manetti,^†

Giuseppe Giannini,*^,‡Loredana Vesci,^‡Ferdinando M. Milazzo,^‡Domenico Alloatti,^‡

Mario B. Guglielmi,^‡Massimo Castorina,^‡Maria L. Cervoni,^‡Marcella Barbarino,^‡Rosanna Fodera,^‡

̀

Valeria Carollo,^‡Claudio Pisano,^‡,⊥Silvia Armaroli,^§and Walter Cabri^‡,#

^†Dipartimento di Biotecnologie, Chimica e Farmacia, Universita

̀

degli Studi di Siena, Via A. Moro 2, I-53100 Siena, Italy

^‡R&D Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina Km 30,400, I-00040, Pomezia, Roma, Italy

^§Sigma-Tau Research Switzerland S.A., Via Motta, 2a, CH-6850 Mendrisio-Stazione, Switzerland

S

* Supporting Information

ABSTRACT: Ruthenium catalyzed 1,3-cycloaddition (click chemistry) of

an azido moiety installed on dihydroxycumene scaﬀold with diﬀerently

substituted aryl propiolates gave a new family of 1,4,5-trisubstituted triazole

carboxylic acid derivatives that showed high aﬃnity toward Hsp90

associated with cell proliferation inhibition, both in nanomolar range.

The 1,5 arrangement of the resorcinol, the aryl moieties, and the pres-

ence of an alkyl (secondary) amide in position 4 of the triazole ring were

essential to get high activity. Docking simulations suggested that the

triazoles penetrate the Hsp90 ATP binding site. Some 1,4,5-trisubstituted

triazole carboxamides induced dramatic depletion of the examined client

proteins and a very strong increase in the expression levels of the

chaperone Hsp70. In vitro metabolic stability and in vivo preliminary studies on selected compounds have shown promising

results comparable to the potent Hsp90 inhibitor NVP-AUY922. One of them, (compound 18, SST0287CL1) was selected for

further investigation as the most promising drug candidate.

agents for human use. Currently, more than 10 Hsp90 inhibitors

are in diﬀerent stages of clinical trials and an impressive growth

in scientiﬁc and patent literature conﬁrms the great interest

toward this pharmacologic target from the academic and the

pharmaceutical industry.¹¹

INTRODUCTION

■

Heat shock protein 90 (Hsp90) is a ubiquitous and abun-

dant ATP-dependent molecular chaperone representing about

1−2% of the whole cytosolic proteomic load,¹increasing up to

∼4−6% under stress conditions.²Hsp90 is a protein highly

conserved from bacteria to mammals, and it is documented to

interact with more than 200 diﬀerent “client” proteins involved

in signal transduction, protein traﬃcking, receptor maturation,

and innate and adaptive immunity.^3,4The main Hsp90 role is

to promote the protein folding and the late-stage of matura-

tion. Most of the so-called oncoproteins are Hsp90 clients.⁴

Consequently, Hsp90 has emerged as an interesting molecular

target for developing new anticancer agents against several solid

and hematological malignancies as well as toward leukemia

stem cells.⁵Moreover, up-regulation of Hsp90 in several solid

tumors likely unveils a “protective” role in tumorigenesis, thus

conﬁrming the key role of this chaperone protein in cancer cell

growth and survival.⁶Besides, recent ﬁndings suggest a

connection between Hsp90 and cytotoxic drug resistance.^7,8

Finally, Hsp90 would also seem to have higher aﬃnity toward

small-molecule inhibitors in tumor cells than in normal cells.⁹

Taken together, all these data point out Hsp90 as an attractive

therapeutic target for cancer.¹⁰Although the target has been

validated using the natural products geldanamycin and radicicol

(Figure 1), to date, there are no FDA approved Hsp90-targeting

Hsp90 inhibitor activity is mainly promoted by a competition

with ATP binding to the N-terminus of the protein, although

some inhibitors act through a noncompetitive mechanism.¹²

Most of the drugs actually in clinical trials are active through

the ATP-competitive mechanism, and some recent reviews

collect most of the information on these molecules.^10,13,14

However, regardless of the chemical scaﬀolds, the new Hsp90

inhibitors have beneﬁted greatly from structure-based design

using available X-ray cocrystal structures¹⁵of diﬀerent

molecules bound to the N-terminal domain of Hsp90,

associated with innovative approaches like high-throughput

screening, fragment-based screening, and virtual screening.¹⁴

Despite the numerous eﬀorts in developing new antitumor

agents with Hsp90 inhibitory properties, the drugs currently

under clinical investigation still show some critical limitations.¹¹

Therefore, a medical need in this ﬁeld is still felt.

Received: October 2, 2013

Published: March 3, 2014

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Figure 1. Some selective Hsp90 inhibitors.

a

In continuation of our interest in discovering new antitumor

compounds,¹⁶⁻¹⁸we explored a new series of potential Hsp90

inhibitors having a 1,2,3-triazole scaﬀold. Although well-known

in medicinal chemistry as a scaﬀold or a linker for diverse bio-

medical applications, 1,2,3-triazoles have never been inves-

tigated as core structures of heterocycles Hsp90 inhibitors. This

fact is even more surprising, since parent heterocycles such as

isoxazole,¹⁹1,2,3-thiadiazole,²⁰or 1,2,4 triazoles^21,22have been

very successful in targeting the ATP binding site of Hsp90.

After a preliminary exploration of the inﬂuence of substituent

arrangement around the heterocycle on the activity as Hsp90

inhibitor, we report here the selective synthesis of various 1,4,5

trisubstituted 1,2,3-triazoles and their complete characterization

as potent Hsp90 inhibitors with promising results in terms of

metabolic stability and in vivo activity. From previous work on

inhibitors of the Hsp90 ATP binding site,¹⁹it is known that the

presence of a resorcinol-like fragment is extremely important to

drive the binding mode and to get a strong interaction with the

enzyme. One of the resorcinol hydroxyl groups displaces one

water molecule required for substrate binding in the ATP

region, while the other phenolic group contributes to a hydro-

gen bond network with the Asp93 residue. Starting from prelim-

inary computational ﬁtting experiments of diﬀerent triazole-

based molecules on the Hsp90 X-ray structure,¹⁸we decided to

keep the resorcinol fragment in position 1 of the triazole and

explore the substitution pattern at positions 4 and 5.

Scheme 1

a

(a) BnBr, K₂CO₃, MeCN followed by MePPh₃Br, BuLi, THF; see ref

19. (b) H₂(1 atm), Pd/C, EtOH, rt, 6 h followed by BnBr, K₂CO₃,

MeCN reﬂux, 3 h (94% overall). (c) HNO₃conc, AcOH, 70 °C,

30 min, (35%). (d) (i) SnCl₂, HCl, EtOH, 80 °C, 3 h; (ii) t-BuONO,

Me₃SiN₃, MeCN, rt, 12 h (75% over two steps).

Alcohols 6 and 7 were therefore transformed into their

corresponding mesylates which were submitted to nucleophilic

displacement with N-phenyl- or N-benzylpiperidine. After the

last removal of the phenolic benzyl protection, compounds 8, 9,

10, and 11 (Scheme 2) were isolated in 65−85% yield and

analyzed in an Hsp90 binding assay. Since the 1,5 disubstituted

regioisomers appeared to be more active compounds than the

corresponding 1,4 disubstitued triazoles (see Table 1), two

alternative fragments were introduced in position 5 following

the same synthetic procedure. Resulting compounds 12 and 13

showed even more encouraging Hsp90 binding properties

in two-digit nanomolar range (see Table 1), conﬁrming that the

1,5 arrangement around the 1,2,3-triazole ring was more pro-

ﬁtable than 1,4 arrangement. However, the activity of com-

pounds 10−13 against a selected panel of cancer cells was poor.

Following experiences previously described in the litera-

ture,^18,19a carboxyamide was introduced in position 4. This

kind of arrangement (1-aryl, 5-aryl, 4-carboxy, 1,2,3-triazole)

has been scarcely studied, and only few examples of regio-

controlled preparation of trisubstituted 1,2,3-triazoles have

been described in the literature.^23,24Alkyne 5 was protected

with TBDMSCl and, after deprotonation with LiHDMS,

coupled with methyl chloroformate to give compound 14

in good yields, the latter being the key intermediate to-

ward the synthesis of the carboxamide variants of 10−13

(Scheme 3).

CHEMISTRY

■

Since 1,2,3-triazoles are suitably prepared by metal catalyzed [3 + 2]

cycloaddition of azides and alkynes, the preparation of arylazide 1

was required. This novel molecule was prepared as described in

Scheme 1, starting from 2,4-dihydroxyacetophenone that was ﬁrst

protected as dibenzyl ether and then transformed¹⁹into alkene 2.

Subsequent reduction followed by an additional step of

benzylation of the phenolic OH (deprotected during reduc-

tion) gave compound 3. The latter was selectively nitrated to

give 4 which was then reduced and treated with TMSN₃in the

presence of tert-butyl nitrite to give the required azide 1 in good

yield (75%). Azide 1 was submitted to Cu(II) catalyzed cyclo-

addition with phenylacetylene derivative 5, giving compound 6

as a single regioisomer although in moderate yields (Scheme 2).

The other regioisomer 7 was obtained via Ru catalysis (Cp*RuCl)₄

in good yields.

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a

of also exploring the inﬂuence of diﬀerent substituents in

position 4, morpholine was introduced on alcohol 16 and the

corresponding methylcarboxylate 36 reacted with diﬀerent

amines to give, after phenolic hydroxyl group deprotection,

amides 37−43 (Scheme 4 and entries 22−28 in Table 2).

Finally, in order to verify the possibility of getting an active

prodrug, compound 18 was acetylated to give 44 (Scheme 4).

Scheme 2

RESULTS AND DISCUSSION

■

We systematically investigated several molecules for their

aﬃnity to human Hsp90 and their antiproliferative activity on

two tumor cell lines (NCI-H460 non-small-cell lung carcinoma

and A431 epidermoid carcinoma cells). As shown in Tables 1

and 2, all the compounds showed appreciable binding to

recombinant human Hsp90α protein in a ﬂuorescence

polarization (FP) assay (see Note S1 in Supporting Information),

with potency similar to or even higher than NVP-AUY922, one

of the most active reference compounds.²⁵In general, tertiary

amines showed better binding than secondary analogues except

in the case of the cyclohexylmethylamine containing derivatives

30 and 31 that retained a binding in the single-digit nanomolar

concentration. Among tertiary amines, better results were

obtained introducing a morpholine fragment (compound 18,

SST0287CL1), also present in the reference product. Analogously,

piperazine derivatives 26 and 27 and the 4-amino substituted

piperidine 28 have a high aﬃnity with Hsp90. It is worth noting

that the piperidine derivative 20 is at least 4 times less active than

the corresponding morpholine or pirrolidine containing com-

pounds 18 and 21. Analogously, the presence of an aliphatic

cycle in position 5 of the triazole is not mandatory for the

activity if a second polar group is present (compare 25 and 18).

Regarding position 4, some variations of the substitution

pattern are tolerated. The introduction of groups with more

than four atoms (38) or with diﬀerent functional group (37)

causes only a partial reduction in the binding (33 and 17 nM,

respectively). A signiﬁcantly lower aﬃnity is found when a

cyclic substituent (39−41) or a branched side chain (42) is

introduced. Although the presence of a substituted carboxamide

is indispensable to get an acceptable level of cytotoxicity, the

inﬂuence of the substituent nature on this property was more

diﬃcult to rationalize. Finally, while acetylation of the OH

prevents the binding to Hsp90 (>1000 nM), the cytotoxicity

of 44 is still comparable with that of the parent compound 18

(6.5 vs 4 nM, respectively), suggesting the possibility to

intervene on this part of the molecule in order to improve the

pharmacokinetic proﬁle of the product.²⁶

The cytotoxicity activity of some compounds of the series

was comparable to that of the reference product NVP-AUY922,

although they showed higher aﬃnity to Hsp90 due to the many

variables associated with a complex system such as a cell. In

particular, 18, 21, and 31 were active toward NCI-H460 cells at

one-digit nanomolar concentration. This antiproliferative

activity trend of 1,2,3-triazole derivatives was conﬁrmed by

results obtained on an extensive panel of tumor cell lines by

means of a sulforhodamine B (SRB) colorimetric test. The

most active derivatives inhibited growth of all tumor cell lines

evaluated irrespective of cancer types or genetics (see Table S1).

Tumor cell lines were selected based on their peculiar genotype,

with particular attention to expression of key wild type or

mutated Hsp90 client proteins or with the aim of covering a wide

array of tumor types (see Table S1) .

a

(a) CuSO₄, Na ascorbate, DMF, rt, 12 h (55%). (b) (Cp*RuCl)₄,

DMF, rt, 3 h (72%). (c) (i) MsCl, TEA, DCM, rt, 2−4 h; (ii) amine,

DMF, Et₃N, rt, 12 h. (d) BCl₃, DCM, 0° C to rt, 2 h.

In fact, the Ru catalyzed cycloaddition of 14 with azide 1

proceeded with complete regioselectivity giving exclusively the

isomer with the -COOMe opposite the residue coming from

the azide (compound 15 in Scheme 3). The coordination be-

tween the carboxylate and the Ru probably makes it the most

hindered group, consequently orienting the transition state

toward compound 15. Removal of the TBDMS group gave

alcohol 16 that, after transformation into the corresponding

mesylate, was submitted to reaction with diﬀerent secondary

amines that gave the expected mesylate displacement. After a

further transformation of the methylcarboxylate in position 4

into ethylamide and removal of the benzylic protection, com-

pounds 18−29 (entries 2−15 in Table 2) were obtained in

good yields. However, when the reaction between the mesylate

derived from 16 and a primary amine was attempted, the

corresponding secondary amines were isolated in very poor

yield. Thus, the procedure was slightly modiﬁed, transforming

ﬁrst the benzyl alcohol 16 into the corresponding benzyl

bromide 17. On this substrate, the nucleophilic displacement

with the primary amines worked well and, after the synthetic

sequence outlined above, compounds 30−35 (entries 16−21 in

Table 2) were isolated in acceptable overall yields. With the aim

In order to have a more precise picture of the interaction

mode of our compounds with Hsp90, docking simulations were

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Table 1. Activity Data of 1,4- or 1,5-Disusbstituted Triazoles

a

Binding to Hsp90 was determined by a ﬂuorescence polarization (FP) assay and cytotoxicity on NCI-H460 non-small-cell lung carcinoma cells.

Data are expressed as IC₅₀mean values ( SD, n = 4) and are in nanomolar concentrations. For the detection limits of the FP assay, see ref 25.

a

The resorcinol hydroxyl group at position 2 shows both

direct and water-mediated hydrogen bonds with the carboxyl

terminus of Asp93. In a similar way, the second resorcinol

hydroxyl group is involved in water-mediated hydrogen bonds

with both the carboxyl terminus of Asp93 and hydroxyl group

of Ser52. A direct hydrogen bond is also found between the

Ser52 OH and the Asp93 carboxyl moiety. The isopropyl group

of the inhibitor is embedded within a large hydrophobic cavity

delimited by Leu48, Leu107, Thr109, Phe138, Val150, and

Val186. The NH and CO portions of the C4 amide group are

anchored by hydrogen bond interactions with the Gly97 carbonyl

and the terminal ammonium group of Lys58, respectively, while

the terminal ethyl chain of the ligand is exposed to solvent and

shows lipophilic contact with the side chain of Ile96 (Figure 2).

A structural motif that signiﬁcantly contributes to the

stability of the complex is constituted by an additional network

of charge-reinforced hydrogen bond interaction involving the

protonable portion of the ligand (i.e., the basic nitrogen atom

of morpholine, piperidine, pyrrolidine, and acyclic amino

groups), the Asp54 carboxy terminus and the ammonium

group of Lys58. The terminal portion of the substituent at C5,

beyond the benzyl moiety and usually corresponding to a

solubilizing functionality (such as morpholine, piperidine,

pyrrolidine, etc.), is pointing toward solvent and does not

show any contact with the protein apart from the interaction

between the protonated nitrogen (if present) and the carboxy

terminus of Asp54. Depending on the terminal substituent, a

diﬀerent number of water molecules can be recruited.²⁷

Scheme 3

a

(a) TBDMSCl, imidazole, DMF, rt, 12 h. (b) LiHDMS, MeOCOCl,

THF, −78 °C (76% over two steps). (c) (Cp*RuCl)₄DMF, rt, 12 h,

(68%). (d) TBAF, THF, rt, 2 h (80%). (e) CBr₄, PPh₃, DCM, rt, 3 h

(87%). (f) MsCl, Et₃N, DCM, 0 °C, then rt for 12 h (90% crude). (g)

Secondary amine, Et₃N, DMF, rt. (h) Ethylamine, MeOH 80 °C, 48 h.

(i) H₂(1 atm), Pd(OH)₂cat., 4 h, rt. (j) Primary amine Et₃N, DMF, rt.

performed using the complex between the N-terminal domain

of Hsp90α and NVP-AUY922 (PDB entry 2VCI) as a template.

Energy minimization of the resulting docked complexes showed

triazole derivatives in an orientation almost identical to that of

the reference inhibitor, with slight diﬀerences in the interaction

pattern, the triazole N2 being involved in a water-bridged

hydrogen bond with the carboxy terminus of Asp93.

An analysis of molecular interactions between Hsp90 and 18

in comparison to those found in the crystallographic complex

with AUY922 does not allow us to justify the diﬀerence in

aﬃnity measured between the two compounds. However,

docking simulations and energy minimization performed on the

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Table 2. Activity Data for 1,4,5-Trisubstituted 1,2,3-Triazole Carboxamides

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Table 2. continued

a

Binding to Hsp90 was determined by a ﬂuorescence polarization (FP) assay and cytotoxicity on NCI-H460 non-small-cell lung carcinoma cells.

Data are expressed as IC₅₀mean values ( SD, n = 4) and are in nanomolar concentrations. For the detection limits of the FP assay, see ref 25.

a

Scheme 4

exposure to several concentrations (dose−response curves) of

selected test compounds, indicated the expected depletion of

the client proteins EGFR, CDK4, and Akt, as well as the

induction of Hsp70 in a dose-dependent manner, thus

conﬁrming the eﬃcacy of these compounds as novel potent

Hsp90 inhibitors (Figures 3 and 4 and Figure S1).

According to the previous data, 18 appeared to have the

most interesting proﬁle, displaying the ability to bind Hsp90

with an aﬃnity comparable to (or even better than)²⁵that of

the reference compound, associated with a strong antiprolifer-

ative activity. Furthermore, 18 caused dramatic depletion of

typical Hsp90 client proteins in A431 cells, associated with a

very strong increase in the expression levels of the chaperone

Hsp70, consistent with inhibition of Hsp90 function (Figure 4).

In addition, as reported in Figure 4, the diacetyl derivative 44

displayed an extremely reduced ability to bind to recombinant

Hsp90α in the FP assay, without signiﬁcant changes in its

antiproliferative eﬃcacy, as a consequence of the Hsp90

chaperone inhibitory property of its putative metabolite (18),

also conﬁrmed by the impressive eﬀect on depletion of client

proteins and on increase of Hsp70 levels.

Flow cytometric analysis revealed that both 18 and NVP-

AUY922 caused cell cycle arrest in G2/M upon treatment of

NCI-H460 NSCLC cells, and a massive induction of apoptosis

was triggered over the recovery times (Figure S2 and Table S2).

Subsequently, the antitumor eﬃcacy of compounds 18 and

19 was evaluated in two tumor xenograft models (A431 epi-

dermoid carcinoma and GTL-16 gastric carcinoma, over-

expressing EGFR and c-Met, respectively). Both compounds,

delivered intraperitoneally for 2 weeks according to the schedule

q2d/wx2w or q4d/wx2w, were able to induce a signiﬁcant

tumor volume inhibition of about 40% (P < 0.05). Moreover,

the compounds were shown to be well-tolerated, since a little

variation of body weight loss was found except when the

molecules were evaluated against GTL-16 which was a

cachectic tumor xenograft (also the vehicle treated group

revealed a signiﬁcant body weight loss) (Table 3). The ref-

erence compound was evaluated at a higher dose according to

the schedule qdx5/wx2w, which was the maximum tolerated

dose to investigate its eﬃcacy (see Figure 5). It is noteworthy

that compounds 18 and 19 were administered at a total dose

6- to 10-fold lower than that used for NVP-AUY922. Its

antitumor eﬃcacy was lower than that found with 18 and 19

a

(a) MsCl, Et₃N, DCM, 0 °C then rt for 12 h. (b) Morpholine, Et₃N,

DMF, rt, 12 h (75% over two steps). (c) (i) LiOH in THF H₂O; (ii)

(COCl)₂; (iii) primary or secondary amine; (iv) BCl₃in DCM, 0°C to

rt (31% over three steps).

Hsp90−18 complex are based on a molecular mechanics

approach (OPLS force ﬁelds) that is, by deﬁnition, almost

unable to account for electronic contribution to binding. A

quantum mechanics treatment of the interactions between

Hsp90 and its inhibitors, which is not among the purposes of

this work, is more appropriate to attempt to justify diﬀerences

in activity. For example, HOMO and LUMO amplitude and

their spatial location as well as dipole moment are signiﬁcantly

diﬀerent in triazoles in comparison to oxazole, thus suggesting a

possibly diﬀerent behavior toward a protein binding site.

The next step was to establish whether the new ligands retain

the well-established molecular signature of known Hsp90

inhibitors in terms of capacity to modulate cellular markers

expression (client proteins). To this aim, the ability of selected

compounds (i.e., 18, 19, 20, 24, 31, 32, 39, and 44) to down-

regulate the expression of representative Hsp90 client proteins

and to induce the expression of Hsp70 protein was determined

in the A431 (human squamous cell carcinoma) cell line

(overexpressing EGFR). Western blot analysis, after 24 h of

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Figure 2. Stereographical representation of the complex between Hsp90 and 18 (ball and stick notation) as derived from molecular docking

calculations and energy minimization (PDB entry 2VCI, 2.0 Å resolution). The triazole core and the resorcinol hydroxyl groups are involved in an

extended network of water-bridged hydrogen bonds. The morpholine solubilizing group is exposed to the solvent and is able to block a water

molecule. For the sake of clarity, only few amino acids are displayed and labeled, together with several water molecules (W). Hydrogen bond

contacts are depicted as black dotted lines.

Figure 3. Analysis of Hsp90 client protein levels (EGFR, Akt, CDK-4) and Hsp70 in A431 tumor cells treated with compound 19 vs17-DMAG and

NVP-AUY922. Total cellular extracts were obtained 24 h after treatment. Actin is shown as a control for protein loading. A representative blot is

shown. Results of densitometry analysis were reported as normalized (to β-actin) ratios. Western blot experiments were performed at least twice

resulting in similar results.

against A431 xenograft and comparable against GTL-16 tumor

xenograft model, as shown in Figure 5.

(EGFR, Akt, and Cdk4) in A431 tumor cell lysates and, at

the same time, signiﬁcantly increased the expression levels of

the chaperone Hsp70, with a potency comparable to that of the

reference compound (NVP-AUY922). Similar results were

observed on the same animal model with compound 19 (data

not shown).

To validate that the in vivo antitumor eﬀect of test com-

pounds was eﬀectively related to the inhibition of Hsp90, the

modulation of selected Hsp90 client proteins was assessed by

Western blot in tumor xenografts a few hours after the last

treatment. As shown in Figure 6, 18 induced a very strong

decrease in the protein levels of three typical client proteins

The human gastric carcinoma cell line GTL-16 is charac-

terized by the overexpression of c-Met (HGF receptor) protein,

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Figure 4. Western blot analysis of Hsp90 client protein levels in A431 tumor cells treated with the lead compound 18 vs its diacetyl prodrug (44).

Analysis was performed as described in Figure 3.

which is another key cellular protein tightly regulated by

Hsp90.^28,29For this reason, c-Met expression was also

evaluated, instead of EGFR, in GTL-16 tumor xenografts in

order to check the in vivo eﬃcacy of 18 and 19. Also, in this

animal model our compounds were shown to potently inhibit

Hsp90, resulting in strong down-regulation of the selected

client proteins and induction of Hsp70, with a similar behavior

compared to the reference compound (Figure 6).³⁰

In addition, our best candidate (18) showed a plasma

stability comparable to that of the reference compound as

shown by a quantitative LC/MS assay performed at 120 min

(65% and 56%, respectively) (Table 4).

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Table 3. Antitumor Activity of 18 Administered ip (with

Schedule q2d/wx2w) and of 19 Given ip (Schedule q4d/

wx2w) on A431 Epidermoid Carcinoma and GTL-16

(Gastric Carcinoma) Cells Implanted Subcutaneously on

a

CD-1 Nude Mice

b

c

d

e

compd

dose (mg/kg)

BWL (%)

lethal toxicity

TVI%

A431 Epidermoid Carcinoma

f

18

19

5

8

1

7

0/8

49

f

45

g

GTL-16 Gastric Carcinoma

f

18

19

5

17

0/8

42

f

42

a

Treatment started 3 days after tumor injection. Eﬃcacy of drugs was

b

evaluated 5 days after the last treatment. Intraperitoneal dose

(mg/(10 mL/kg)) used in each administration. Maximum BWL

percentage due to the drug treatment. Dead/treated animals. TVI%

versus control mice. P < 0.05 vs vehicle-treated group (Mann−

Whitney test). Tumor xenograft induced cachexia.

c

d

e

f

g

Finally, starting from the observation that a long chain

residue installed on the amide did not signiﬁcantly reduce the

activity, a new derivative with a hydroxamic acid group on

the hexamethylene chain was synthesized (compound 43 in

Table 2). This group is a zinc binding group widely used in

histone deacetylase (HDAC) inhibitors to coordinate the Zn

ion located within the enzymatic site. The introduction of the

HDAC binding arm fragment on a chemical structure already

active on HSP90 did not reduce the binding activity too much

(31 nM), although it was associated with a reduction of the

cytotoxic activity (>1 μM). On the other hand, 43 was also

investigated as HDAC inhibitor on a panel of 10 isolated

human HDAC isozymes (see Supporting Information). The

assays were done in the presence of a ﬂuorogenic peptide

bound to the RHKK(Ac) fragment of p53 (residues 379−392),

as the substrate, and using SAHA as the reference compound

(Table 5).³¹Surprisingly, in addition to a good aﬃnity to

Hsp90, 43 also demonstrated a high selectivity toward the

HDAC6 isoform. HDAC6 is predominantly a cytoplasmic,

microtubule-associated member of the class IIB HDAC family,

which is directly involved in controlling the HSP90 acetylation

degree and function. Depletion of HDAC6 activity has been

reported to aﬀect HSP90 function in tumor cells.³²These

preliminary data support 43 as an interesting lead compound

for further studies in the dual targeting HDAC/Hsp90

inhibition approach.

CONCLUSIONS

■

Among diﬀerent 1,4,5-trisubstituted 1,2,3-triazole carboxamides

prepared by regioselective Ru-catalyzed Huisgen reaction, the

triazole scaﬀold carrying a resorcinol moiety in position 1, an

ethylcarboxamide in position 4, and an arylmethylamine in

position 5 were demonstrated to have nanomolar binding to

the N-terminus of Hsp90 and to induce cell death in diﬀerent

tumor cell lines. The antitumor activity of this class of

compounds was demonstrated to proceed through Hsp90

inhibition, justiﬁed by a dramatic decrease in the levels of three

typical client proteins (EGFR, Akt, and Cdk4) in A431 cell

lysates and, at the same time, a strong increment of expression

levels of the chaperone Hsp70. Results of ﬂuorescence activated

cell sorting analysis showed that compound 18 caused cell cycle

arrest in G2/M upon treatment of NCI-H460 NSCLC cells and

that a massive induction of apoptosis was triggered over the

Figure 5. Antitumor eﬃcacy of compounds 18 and 19 delivered

intraperitoneally in comparison with NVP-AUY922 against A431

epidermoid carcinoma (overexpressing EGF receptor) (A, B) and

GTL-16 gastric carcinoma (overepressing c-Met) (C), xenografted sc

in nude mice.

recovery times. Several cancer cell lines were sensitive to

diﬀerent members of the class. Moreover, results of in vivo

experiments carried out on diﬀerent tumor xenograft models in

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to build new Hsp90 inhibitors (including potential HDAC/

Hsp90 dual inhibitors). Further investigations are in progress to

optimize the dose−activity proﬁle of the compounds and to

investigate this class of molecules in other therapeutic areas

related to Hsp90 inhibition.

EXPERIMENTAL SECTION

■

Animals. In vivo experiments were carried out using female

athymic nude mice, 5−6 weeks old (Harlan). Mice were maintained in

laminar ﬂow rooms, keeping temperature and humidity constant. Mice

had free access to food and water. Experiments were approved by the

Ethics Committee for Animal Experimentation of Sigma-Tau

according to institutional guidelines.

Xenograft Tumor Model. For subcutaneous (sc) tumor model,

exponentially growing tumor cells (5 × 10⁶/100 μL) were sc in-

oculated in the right ﬂank of nude mice. Mice were treated starting

3 days after tumor injection. Tumor volume (TV) was measured

biweekly, and it was calculated according to the formula TV (mm³) =

d²D/2, where d and D are the shortest and the longest diameter,

respectively. The selected molecules were delivered intraperitoneally

according to the schedule q2d/wx2w or q4d/wx2w. Drug eﬃcacy was

assessed as (i) tumor volume inhibition percentage (TVI%) in drug-

treated versus control mice, expressed as TVI% = 100 − [(mean TV

treated/mean TV control) × 100]. The days of TVI evaluation are

reported in the Table 3. Toxic eﬀects of the drug treatment were

assessed as the following: (i) body weight loss percentage (BWL%),

calculated as BWL% = 100 − [(mean BW day x/mean BW day 1) ×

100], where day 1 was the ﬁrst day of treatment and day x was any day

thereafter; the highest (max) BWL% is reported in Table 3; (ii) lethal

toxicity, assessed as deaths occurring in treated mice before the death

of the ﬁrst control mouse.

The in vivo eﬀect of selected inhibitors on the expression of typical

Hsp90 client proteins was carried out by Western blotting, as

described in the section Client Protein Degradation Assay, on whole

protein lysates that were prepared through homogenization of tumor

samples (excised 2 or 6 h after the last treatment) in T-PER tissue

protein extraction reagent (Pierce, Rockland, IL, U.S.), supplemented

with 10 μg/mL protease inhibitor cocktail (Sigma Chemical Co., St.

Louis, MO, U.S.).

Cell Lines and Culture Conditions. The A431 epithelial

carcinoma cell line (ATCC), the NCI-H460 non-small-cell lung

carcinoma cell line (ATCC), the ovarian carcinoma cell line A2780

(ECACC), the MDA-MB436 breast carcinoma cell line (ATCC), the

HeLa cervix uteri carcinoma cell line (Istituto Zooproﬁlattico di

Brescia, Italy), and the NB4 promyelocytic leukemia cell line (DSMZ)

were routinely grown in RPMI-1640 medium (Lonza, Vierviers,

Belgium), supplemented with 10% (v/v) heat-inactivated fetal bovine

serum (GIBCO, Invitrogen, Paisley, U.K.). The GTL-16 gastric

carcinoma cell line (kindly provided by Metheresis Translational

Research S.A.) and the MiaPaCa2 pancreas carcinoma cell line

(ECACC) were grown in DMEM (Lonza, Vierviers, Belgium),

Figure 6. Analysis of Hsp90 client protein levels (EGFR, Akt, CDK-4,

c-Met) and Hsp70 in A431 (A) and GTL-16 (B) tumor xenografts,

following treatment with 18 and 19, with respect to the reference

compound (NVP-AUY922). Total proteins were puriﬁed 2 h (in the

case of A431 tumors) or 6 h (in the case of GTL-16 tumors) after the

last treatment. Actin is shown as a control for protein loading.

Representative blots of two tumor samples/group are shown.

Table 4. Plasma Stability of Lead Compound 18 vs

a

Reference Compound

compd

plasma stability (%) at 120 min

NVP-AUY922

65.5 1.6

56.1 2.2

18

a

Data represent the mean SD of a representative experiment.

mice showed that 18 (SST0287CL1) induces a signiﬁcant

tumor volume inhibition and is well-tolerated, being active at a

lower dose with respect to the reference compound. The

diacetate derivative of the most active compound, maintaining a

nanomolar cytotoxicity, suggests the possibility of modifying

this region of the structure to improve pharmacokinetic prop-

erties and bioavailability. These results point out that easily

aﬀordable 1,4,5-trisubstituted 1,2,3-triazoles are useful scaﬀolds

Table 5. Diﬀerent HDAC Isoform Inhibitory Activity (IC₅₀, μM) of 43, an HDAC/Hsp90 Dual Inhibitor, Compared with

Suberoylamide Hydroxamic Acid (SAHA)

HDAC isoform

1

2

3

4

5

6

7

8

9

10

11

43

3.10

0.26

5.61

0.92

2.79

0.35

71.50

0.493

1.91

0.012

0.029

45.50

0.344

0.717

0.243

3.22

1.77

SAHA (vorinostat)

0.378

0.316

0.456

0.362

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supplemented with 10% (v/v) heat-inactivated fetal bovine serum

(GIBCO). The U87MG glioblastoma cell line (ATCC), the A498

renal carcinoma cell line (ATCC), and the MeWo melanoma cell line

(ECACC) were grown in EMEM (Lonza, Vierviers, Belgium),

supplemented with 10% (v/v) heat-inactivated fetal bovine serum

(GIBCO). The HCT-116 colon carcinoma cell line (Istituto

Zooproﬁlattico di Brescia) was grown in McCoy’s 5A medium

(Lonza, Vierviers, Belgium), supplemented with 10% (v/v) heat-

inactivated fetal bovine serum (GIBCO).

Plasma Stability. Test compounds were added to human plasma

to get a ﬁnal concentration of 5 μM and incubated at 37 °C at diﬀerent

times (until 120 min). Then 0.1 mL of each sample was taken, added

in a corresponding 2-fold amount of chilled acetonitrile, centrifuged at

+4 °C. The supernatant was then submitted to LC−MS/MS analysis.

At the end of incubation, the % of residual intact compound remaining

was calculated.

Docking. The three-dimensional structure of Hsp90 in complex

with a 4,5-diarylisoxazole inhibitor derived from X-ray crystallography

(PDB entry 2VCI, 2.0 Å resolution)¹⁹was used to create the initial

coordinates for docking calculations. Protein Preparation Wizard

(implemented within Maestro 9.2, Schroedinger, LCC) was used to

prepare the protein structure, also adding hydrogen atoms and

sampling water orientation. HOH2232, HOH2099, HOH2233, and

HOH2115, which constitute a network of ordered and conserved

water molecules, were kept for next calculations.

For preliminary ﬁtting calculations of diﬀerent triazole-based

molecules on the Hsp90 ATP binding site,¹⁸inhibitor structures

were prepared with LigPrep routine (implemented within Maestro 9.2,

Schroedinger, LCC). Their conformations were generated by means of

MacroModel, version 9.9 (Monte Carlo multiple minimum algorithm

and an energy window of 10 kcal/mol), and docking calculations were

performed with Glide (version 5.6). Final rescoring of best poses was

carried out with Prime 2.2, MM-GBSA routine.

Since Glide only modiﬁes the torsional internal coordinates of the

ligand during docking, without taking into account the remaining

geometric parameters, we chose to apply a conformational search/

energy minimization protocol to study the interaction between Hsp90

and the new triazole inhibitors. In particular, the structure of the

cocrystallized isoxazole inhibitor was replaced by that of triazole

derivatives (built with LigPrep) and the resulting complexes were

submitted to a statistical conformational search (10 000 steps of

statistical pseudo Monte Carlo algorithm), sampling all the rotatable

bonds of the inhibitors. Moreover, the overall structure of the ligand

was allowed to rotate and translate within the Hsp90 binding site. A

continuum solvation approach was also applied, with the OPLS_2005

force ﬁelds and the Polak−Ribiere conjugate gradient algorithm for

energy minimization (derivative convergence 0.01 kcal Å⁻¹mol⁻¹).

Only a substructure constituted by the inhibitor and a shell of amino

acids within a 5 Å radius from the inhibitor was submitted to energy

minimization while keeping ﬁxed the remaining part of the complex.

Chemistry. Reagents were purchased from commercial suppliers

and used without further puriﬁcation. ¹H NMR spectra were recorded

at the ﬁeld frequency and in the solvent indicated. Peak positions are

given in parts per million downﬁeld from tetramethylsilane as the

internal standard. J values are expressed in hertz. LC−MS data were

recorded on a Waters Micromass ZQ-2000 instrument or on a double-

focusing Finnigan MAT 95 instrument with BE geometry. Thin layer

chromatography (TLC) was carried out using Merck precoated silica

gel F-254 plates. Flash chromatography was done using Merck silica

gel 60 (0.063−0.200 mm), using light petroleum (bp 40−60 °C) PE.

Solvents were dried according to standard procedures, and reactions

requiring neutral atmosphere conditions were performed under argon.

Solutions containing the ﬁnal products were dried with Na₂SO₄,

ﬁltered, and concentrated under reduced pressure using a rotatory

evaporator. All of the ﬁnal products undergoing biological testing were

>95% pure as demonstrated by analysis carried out with a Varian

Prostar HPLC system equipped with an UV variable wavelength

detector at 254 nm (column Gemini-NX C18 (150 mm × 4.8 mm,

gradient A/B 95/5 to 50/50 in 15 min, A consisting of 0.1% formic

acid in H₂O, B consisting of 0.1% formic acid in acetonitrile, ﬂow rate

of 1.5 mL/min, room temperature).

Cellular Sensitivity to Drugs. NCI-H460 non-small-cell lung

carcinoma and A431 epidermoid carcinoma cell lines were grown in a

volume of 200 μL at approximately 10% conﬂuence in 96-well-plates

and were allowed to recover for an additional 24 h. Tumor cells were

treated with either varying concentrations of drugs or solvent for 72 h.

The fraction of cells surviving after compound treatment was

determined using the SRB assay. IC₅₀values were deﬁned as the

drug concentration causing a 50% reduction in cell number compared

with that of vehicle-treated cells and evaluated by the “ALLFIT”

computer program by analyzing dose−response inhibition curves.

Binding on Hsp90 by a Fluorescence Polarization (FP)

Assay. The ability of test compounds to compete with a ﬂuorescently

labeled probe for binding to full-length recombinant human Hsp90α

(catalog no. SPP-776 Stressgene, Victoria, BC, Canada) was

determined by means of a suited ﬂuorescence polarization assay, as

previously described.¹⁸GM-FITC, supplied by Invivogen (06C23-MT,

CA, USA), was used as probe. Fluorescence polarization (FP)

measurements were performed on a multilabel reader (Wallac

Envision 2101, Perkin Elmer, Zaventem, Belgium). Measurements

were taken in Opti-Plate-96F well plates (Perkin Elmer, Zaventem,

Belgium). The compounds were previously dissolved in DMSO and

kept at −20 °C until use. On the day of experiment, serial dilutions of

stock solutions were prepared in assay buﬀer (HFB) containing

20 mM HEPES, pH 7.3, 50 mM KCl, 5 mM MgCl₂, 20 mM Na₂MoO₄,

and 0.01% NP40. Bovine γ globulin (0.1 mg/mL) and 2 mM DTT

were freshly added to the assay buﬀer immediately before use. Each

assay was carried out by adding 50 μL of the GM-FTC solution

(5 nM) to 30 nM of Hsp90 in the presence of 5 μL of the test com-

pounds at increasing concentrations. The plates were heated at 4 °C

for 4 h, and then the FP values in mP (millipolarization unit) were

recorded. The IC₅₀values were calculated as the inhibitor concentration

allowing displacement of 50% of the tracer, through nonlinear least-

squares analyses and curve ﬁtting using the Prism GraphPad software

program (GraphPad Software, Inc., San Diego, CA).

Client Protein Degradation Assay. Client protein degradation

was determined by Western blotting as previously described.¹⁸

Twenty-four hours after seeding, A431 (human epidermoid

carcinoma) cells were treated for 24 h with test compounds at various

concentrations (dose−response curve), depending on their relative

potency, and then processed to obtain whole-cell extracts. 17-DMAG

(at a concentration of 0.2 μM) was used as internal reference inhibitor.

Following treatment, cells were rinsed twice with ice-cold PBS and

then lysed in RIPA buﬀer supplemented with protease and phos-

phatase inhibitors. Protein concentration was determined by the

Bradford protein assay (Thermo Scientiﬁc, Rockford, IL, USA). Equal

amounts of total proteins were separated through SDS−PAGE and

then blotted onto nitrocellulose membranes. Nonspeciﬁc binding sites

were blocked by incubation of the membranes with 5% nonfat dry

milk in TBS, overnight at 4 °C. Membranes were ﬁnally probed with

the following primary antibodies: anti-EGFR (Upstate Biotechnology,

Millipore Corporate, Billerica, MA, USA), anti-Cdk4 (Santa Cruz

Biotechnology Inc., CA, USA), anti-Akt (Cell Signaling Technology,

Inc., MA, USA), anti-HSP70 (BRM-22), and anti-actin (Sigma

Chemical Co., St. Louis, MO, USA). After extensive washings in

TBS-T, membranes were incubated with appropriate dilutions of a

horseradish peroxidase conjugated corresponding secondary antibody.

Finally, immunoreactive bands were detected by means of the ECL

Plus detection system (GE Healthcare Bio-Sciences, Uppsala, Sweden)

and acquired by a phosphorimaging system (STORM 860; Molecular

Dynamics, Sunnyvale, CA).

2,4-Bis(benzyloxy)cumene (3). Pd/C (223 mg, 0.6 mmol) was

added to a solution of 2 (4 g, 12.11 mmol)¹⁷in ethanol (40 mL) at

room temperature, and the mixture was hydrogenated (balloon, 1 atm)

for 12 h. The catalyst was ﬁltered out through Celite, and the ethanol

was removed under reduced pressure. The residue was dissolved in

CH₃CN (25 mL), and K₂CO₃(7.00 g, 50.6 mmol) was added. The

suspension was stirred for 15 min at room temperature. Benzyl

bromide (5.49 mL, 50.6 mmol) was then added dropwise, and the

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reaction mixture was reﬂuxed for 3 h. H₂O (40 mL) was added, and

the organic phase was extracted with EtOAc (3 × 30 mL). The organic

phase was dried (Na₂SO₄) and the solvent removed. The title com-

pound was obtained as a colorless solid (3.8 g, 94%). Mp 76−78 °C.

¹H NMR (400 MHz, CDCl₃): δ 7.45−7.31 (m, 10 H), 7.11 (d, J = 8.4

Hz, 1 H), 6.60−6.53 (m, 2 H), 5.04 (s, 2 H), 5.02 (s, 2 H), 3.41−3.25

(m, 1 H), 1.21 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100 MHz, CDCl₃): δ

157.9, 157.7, 136.6, 136.0, 129.2, 128.5, 128.4, 128.3, 127.9, 127.1,

126.6, 108.3, 102.3, 70.3, 70.0, 27.1, 23.3.

136.09, 131.75, 130.62, 128.61, 128.37, 128.04, 127.83, 127.62, 127.11,

126.95, 126.69, 126.48, 125.84, 118.56, 99.08, 70.71, 70.31, 64.60,

26.44, 22.57.

4-(5-{p-[(4-Phenyl-1-piperazinyl)methyl]phenyl}-1H-1,2,3-

triazol-1-yl)-1,3-cumenediol (10). General Procedure. Et₃N (160 μL,

1.17 mmol) and MsCl (90 μL, 1.17 mmol) were added to a solution of

7 (200 mg, 0.39 mmol) in DCM (5 mL) at 0 °C. The solution was

stirred for 30 min at 0 °C and for 12 h at room temperature. The

solvent was removed under reduced pressure, and the reaction crude

was dissolved in DMF (2−3 mL). 1-Phenylpiperazine (189 mg, 1.17

mmol) and Et₃N (160 μL, 1.17 mmol) were added, and the mixture

was stirred for 12 h at room temperature. The mixture was diluted

with H₂O and EtOAc, and the organic phase was extracted with

EtOAc (4 × 30 mL) and washed with H₂O (2 × 30 mL) and with

brine (2 × 30 mL). The solvent was evaporated, and the reaction

crude was puriﬁed by passing through a silica gel cartridge, eluting with

EtOAc. The fraction was collected, and the solvent was removed. The

residue obtained was dissolved in dry DCM (3 mL), cooled to 0 °C,

and BCl₃(680 μL of a solution 1 M in DCM, 0.68 mmol) was added.

The mixture was stirred for 2 h at room temperature. Saturated

aqueous solution of NaHCO₃was added (until the pH became slightly

basic), and the organic phase was extracted with DCM (3 × 10 mL),

washed with H₂O, and dried over Na₂SO₄. The solvent was removed

under reduced pressure. The reaction crude was puriﬁed by column

chromatography (DCM/MeOH, 90/10), and the title compound was

obtained as a purple oil (157 mg 86%). ¹H NMR (400 MHz, DMSO-

d₆): δ 10.25 (bs, 1 H), 10.09 (bs, 1 H), 9.25 (s, 1 H), 7.44−7.35 (m, 6

H), 7.20−7.16 (m, 2 H), 6.90−6.88 (m, 2 H), 6.77−6.73 (m, 1 H),

6.51 (s, 1 H), 3.52 (s, 2 H), 3.15−3.07 (m, 5 H), 2.49−2.47 (m, 4 H),

1.09 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100 MHz, DMSO-d₆): δ 150.2,

150.1, 149.9, 144.3, 138.5, 131.2, 130.1, 129.1, 127.0, 125.7, 122.2,

120.4, 120.3, 116.8, 105.6, 62.9, 53.7, 50.7, 25.3, 22.7. HRMS (ESI)

calcd for C₂₈H₃₂N₅O₂(M + 1)⁺470.2556; found 470.2554.

1- Nitro-2,4-bis(benzyloxy)cumene(4). HNO₃(1.11 mL, 13.73

mmol) was added to a suspension of 3 (3.8 g, 11.44 mmol) in AcOH

(46 mL), and the mixture was heated at 70 °C for 30 min. The

solution was cooled to 0 °C and neutralized with aqueous NaHCO₃.

The organic phase was extracted with EtOAc, and the solvent was

removed under reduced pressure. The crude was puriﬁed by ﬂash

chromatography (PE/EtOAc, 95/5 to 90/10), and the title compound

was obtained as a yellow solid (1.51 g, 35%). Mp: 89−90 °C. ¹H NMR

(400 MHz, CDCl₃): δ 7.87 (s, 1 H), 7.43−7.31 (m, 10), 6.52 (s, 1 H),

5.14 (s, 2 H), 5.05 (s, 2 H), 3.30−3.23 (m, 1 H), 1.20 (d, J = 7.2 Hz, 6

H). ¹³C NMR (100 MHz, CDCl₃): δ 160.1, 156.8, 138.3, 136.6, 134.8,

129.1, 128.4, 127.9, 126.6, 125.9, 124.6, 98.7, 72.1, 70.3, 26.5, 22.8.

1- Azido-2,4-bis(benzyloxy)cumene (1). SnCl₂·2 H₂O (5.38 g,

23.85 mmol) and HCl (4.7 mL, 56.85 mmol) were added to a

suspension of 4 (1.5 g, 3.98 mmol) in EtOH (38 mL). The mixture

was heated at 80 °C for 3−4 h. Then it was cooled to 0 °C and NaOH

(28 mL of a 20% water solution) was added. The separated salts were

ﬁltered through Celite and washed with EtOAc. The organic phase was

extracted three times with EtOAc, and the solvent was evaporated. To

the residue (1.10 g, 3.18 mmol, 80%) dissolved in CH₃CN (40 mL)

were added t-BuONO (2.25 mL, 19 mmol) and TMSN₃(2 mL, 15.2

mmol), and the mixture was stirred for 1 h at 0 °C and for 12 h at

room temperature. The solvent was evaporated, and the reaction crude

was puriﬁed by ﬂash chromatography (PE/EtOAc, 95/5 to 90/10).

The title compound was obtained as a brown waxy material (890 mg,

75%). ¹H NMR (400 MHz, CDCl₃): δ 7.40−7.31 (m, 10 H), 6.82 (s,

1 H), 6.52 (s, 1 H), 5.04 (s, 2 H), 4.96 (s, 2 H), 3.31−3.24 (m, 1 H),

1.16 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100 MHz, CDCl₃): δ 157.8,

150.2, 139.3, 136.6, 132.6, 128.4, 127.8, 126.6, 125.6, 124.8, 117.7,

101.8, 70.3, 70.1, 26.3, 22.7. HRMS (ESI) calcd for C₂₃H₂₃N₃O₂Na

(M + Na)⁺396.1688; found 396.1687.

4-(4-{p-[(4-Phenyl-1-piperazinyl)methyl]phenyl}-1H-1,2,3-

triazol-1-yl)-1,3-cumenediol (8). Column chromatography was

carried out with (DCM/MeOH, 90/10). The title compound was

1

obtained as a dense gray oil (110 mg, 60%). H NMR (400 MHz,

DMSO-d₆): δ 8.03 (bs, 2H), 7.84−7.04 (m, 6H), 7.06−6.61 (m, 4H),

6.67−6.19 (m, 2H), 3.52 (s, 2H), 3.46−2.88 (m, 5H), 2.83−2.12 (m,

4H), 1.07 (d, J = 6.8 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ

155.7, 151.8, 151.2, 150.2, 140.3, 129.1, 128.1, 126.7, 126.4, 124.1,

122.1, 120.3, 120.0, 116.8, 113.5, 100.5, 62.8, 56.7, 51.4, 23.3, 20.7.

HRMS (ESI) calcd for C₂₈H₃₂N₅O₂(M + 1)⁺470.2556; found

470.2553.

1-[2,4-Bis(benzyloxy)cumenyl]-4-[p-(hydroxymethyl)-

phenyl]-1H-1,2,3-triazole (6). Azide 1 (100 mg, 0.27 mmol) was

dissolved in DMF (1.5 mL) followed by (p-ethynylphenyl)methanol 5

(35 mg, 0.27 mmol), sodium ascorbate (5 mg, 0.027 mmol), and

CuSO₄·5H₂O (0.67 mg, 0.0027 mmol). The mixture was stirred at

room temperature for 12 h. Water and EtOAc were added. The

organic layer was separated, dried over anhydrous MgSO₄and the

solvent evaporated. Column chromatography on silica gel (PE/EtOAc,

4-(4-{p-[(4-Benzyl-1-piperazinyl)methyl]phenyl}-1H-1,2,3-tri-

azol-1-yl)-1,3-cumenediol (9). Column chromatography was

carried out with (DCM/MeOH, 90/10). The title compound was

1

obtained as a purple oil (97 mg, 52%). H NMR (400 MHz, DMSO-

1

d₆): δ 8.13 (bs, 2H), 7.77−7.06 (m, 10H), 6.56 (m 1H), 6.40 (s, 1H),

3.52 (s, 4H), 3.22(q, J = 7 Hz, 1H), 2.81−2.18 (m, 8H), 1.11 (d, J = 7

Hz, 6 H). ¹³C NMR (100 MHz, DMSO-d₆): δ 155.7, 151.8, 151.2,

140.6, 137.8, 129.2, 128.1, 128.1, 127.5, 126.7, 126.4, 124.1, 122.1,

120.0, 113.5, 100.5, 62.9, 62.7, 52.8, 51.5, 25.7, 21.4. HRMS (ESI)

calcd for C₂₉H₃₄N₅O₂(M + 1)⁺484.2713; found 484.2711.

4-(5-{p-[(4-Benzyl-1-piperazinyl)methyl]phenyl}-1H-1,2,3-tri-

azol-1-yl)-1,3-cumenediol (11). Column chromatography was

carried out with (DCM/MeOH, 90/10). The title compound was

obtained as a purple oil (158 mg, 85%). ¹H NMR (400 MHz,

DMSO): δ 10.26 (bs, 1 H), 10.11 (bs, 1 H), 9.24 (s, 1 H), 7.43−7.29

(m, 10 H), 6.54 (s, 1 H), 3.48−3.31 (m, 6 H), 3.13−3.06 (m, 1 H),

2.42−2.32 (m, 6 H), 1.08 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100 MHz,

DMSO): δ 150.1, 149.9, 144.3, 138.7, 137.8, 131.1, 130.1, 129.2, 128.1,

127.5, 127.0, 127.0, 125.7, 122.2, 120.4, 105.6, 62.9, 62.7, 52.8, 25.3,

22.7. HRMS (ESI) calcd for C₂₉H₃₄N₅O₂(M + 1)⁺484.2713; found

484.2715.

60/40) gave compound 6 as a waxy material (76 mg, 55%). H NMR

(400 MHz, CDCl₃): δ 7.63 (m, 2H), 7.40−7.22 (m, 12 H), 6.66 (s,

1H), 5.08 (s, 2H), 5.05 (s, 2H), 4.72 (s, 2H), 3.35 (qn, J = 7 Hz, 1 H),

2.25 (bs, 1H), 1.23 (d, 6 H, J = 7 Hz). ¹³C NMR (100 MHz, CDCl₃):

δ 160.5, 156.4, 151.8, 142.5, 136.0, 135.9, 135.2, 128.7, 128.4, 128.0,

127.8, 127.6, 127.2, 126.5, 124.4, 123.8, 120.0, 112.1, 102.6, 70.1, 70.1, 64.2.

1-[2,4-Bis(benzyloxy)cumenyl]-5-[p-(hydroxymethyl)-

phenyl]-1H-1,2,3-triazole (7). Azide 1 (890 mg, 2.38 mmol) was

dissolved in DMF (5 mL), and (p-ethynylphenyl)methanol 5 (286 mg,

2.16 mmol) was added at room temperature. The ﬂask was subjected

to three vacuum−nitrogen cycles. Then [Cp*RuCl]₄(116 mg, 0.11

mmol) was added followed by other three vacuum−nitrogen cycles.

The mixture was stirred at room temperature for 6 h. EtOAc (10 mL)

and water (20 mL) were then added. The organic phase was extracted

four times with EtOAc (10 mL), washed with water (three times) and

brine (one time), and dried over Na₂SO₄. The solvent was removed,

and the mixture was puriﬁed by column chromatography (PE/EtOAc,

60/40). The title compound was obtained as a purple oil (866 mg,

4-(5-{p-[(1-Morpholyl)methyl]phenyl}-1H-1,2,3-triazol-1-yl)-

1,3-cumenediol (12). Column chromatography was carried out with

(DCM/MeOH, 95/5). The title compound was obtained as a purple

oil (105 mg. 68%). ¹H NMR (400 MHz, MeOD): δ 8.92 (s, 1 H), 7.47

(s, 4 H), 7.20 (s, 1 H), 6.38 (s, 1 H), 4.90 (bs, 2 H), 3.82 (s, 2 H),

1

72%). H NMR (400 MHz, CDCl₃): δ 7.85 (s, 1 H), 7.36−7.14 (m,

13 H), 6.87−6.83 (m, 2 H), 6.45 (s, 1 H), 4.97 (s, 2 H), 4.71 (s, 2 H),

4.69 (s, 2 H), 3.32 (qn, J = 6.8 Hz, 1 H), 1.21 (d, 6 H, J = 6.8 Hz). ¹³C

NMR (100 MHz, CDCl₃): δ 157.65, 151.46, 141.66, 139.27, 136.42,

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3.75−3.68 (m, 4 H), 3.25−3.12 (m, 1 H), 2.78−2.70 (m, 4 H), 1.15

(d, J = 6.8 Hz, 6 H). ¹³C NMR (100 MHz, MeOD): δ 150.1, 149.9,

144.3, 138.0, 131.1, 130.1, 127.1, 126.8, 125.7, 122.2, 120.4,

105.6, 67.1, 63.2, 53.8, 25.3, 22.7. HRMS (ESI) calcd for

C₂₂H₂₇N₄O₃(M + 1)⁺395.2083; found 395.2080.

4-(5-{p-[(3-Hydroxy-1-piperazinyl)methyl]phenyl}-1H-1,2,3-

triazol-1-yl)-1,3-cumenediol (13). Column chromatography was

carried out with (DCM/MeOH, 90/10). The title compound was

obtained as a purple oil (105 mg, 66%). ¹H NMR (400 MHz, MeOD):

δ 8.93 (s, 1 H), 7.49 (s, 4 H), 7.21 (s, 1 H), 6.40 (s, 1 H), 4.88 (bs,

3 H), 3.90 (s, 2 H), 3.92−3.77 (m, 1 H), 3.24−3.12 (m, 1 H), 2.95−

2.50 (m, 4 H), 1.90−1.40 (m, 4 H), 1.16 (d, J = 6.8 Hz, 6 H). ¹³C

NMR (100 MHz, MeOD): δ 150.1, 149.9, 144.3, 139.3, 131.1, 130.1,

127.2, 126.6, 125.7, 122.2, 120.4, 105.6, 66.9, 66.8, 61.6, 55.4, 33.3,

25.3, 23.3, 22.7. HRMS (ESI) calcd for C₂₃H₂₉N₄O₃(M + 1)⁺

409.2240; found 409.2237.

127.63, 126.78, 126.45, 125.74, 125.62, 124.30, 116.98, 98.40, 70.33,

69.91, 63.87, 51.66, 26.04, 22.20. HRMS (ESI) calcd for

C₃₄H₃₃N₃O₅Na (M + Na)⁺587.2352 (36.8%) 586.2318 (100%);

found 587.2350 (36.6%) 586.2316 (100%).

Methyl 1-[2,4-Bis(benzyloxy)cumenyl]-5-[p-(bromomethyl)-

phenyl]-1H-1,2,3-triazole-4-carboxylate (17). Triphenylphos-

phine (1.45 g, 5.54 mmol) and carbon tetrabromide (1.84 g,

5.54 mmol) were added to a solution of alcohol 16 (2.4 g, 4.26

mmol) in dry DCM (48 mL) at 0 °C. The mixture was stirred at this

temperature for 1 h. Then the solvent was removed under reduced

pressure and the residue was puriﬁed by column chromatography

(PE/EtOAc, 70/30). The title compound was obtained as a purple oil

(2.32 g, 87%). However, the crude product could be directly employed

1

in the next step without any chromatographic puriﬁcation. H NMR

(400 MHz, CDCl₃): δ 7.40−7.16 (m, 13 H), 7.08−6.99 (m, 2 H), 6.41

(s, 1 H), 4.96 (s, 2 H), 4.71 (s, 2 H), 4.44 (s, 2 H), 3.89 (s, 3 H),

3.32−3.20 (m, 1 H), 1.16 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100 MHz,

CDCl₃): δ 161.2, 157.5, 151.0, 141.8, 138.6, 135.9, 135.6, 135.2, 130.1,

129.7, 128.6, 128.1, 127.6, 126.7, 126.4, 125.7, 125.5, 117.0, 98.5, 70.4,

69.9, 51.6, 32.3, 26.0, 22.1. HRMS (ESI) calcd for C₃₄H₃₂BrN₃O₄Na

(M + Na)⁺650.1454 (97.5%), 648.1474 (100%); found 650.1452

(97%), 648.1471 (100%).

Methyl 3-[p-(tert-Butyldimethylsilyloxymethyl)phenyl]-

propiolate (14). LiHMDS (5.8 mL of a solution 1 M in toluene,

5.8 mmol) was added to a solution of the tert-butyldimethylsilyl ether

of 5 (820 mg, 3.33 mmol) in dry THF (28 mL) at −78 °C. The

reaction mixture was slowly warmed to −40 °C and left for 1 h at this

temperature. ClCOOMe (420 μL, 5.41 mmol) was dissolved in THF

(9.6 mL) and cooled to −40 °C. The mixture prepared before was

added to this solution via cannula, and the mixture was warmed to

room temperature. A saturated solution of NH₄Cl was added to the

mixture reaction. The organic phase was extracted with EtOAc (3 ×

20 mL) and dried. The solvent was removed under reduced pressure,

and the reaction crude was puriﬁed by column chromatography

(PE/EtOAc, 95:5). The title compound was obtained as a yellow oil

{1-[2,4-Dihydroxycumenyl]-5-[p-(4-morpholin-4-ylmethy)-

phenyl]-1H-1,2,3-triazol-4-yl}(ethylamino)formaldehyde (18).

General Procedure. Et₃N (230 μL, 1.65 mmol) and MsCl (130 μL,

1.65 mmol) were added to a solution of 16 (310 mg, 0.55 mmol) in

DCM (7 mL) at 0 °C. The solution was stirred for 30 min at 0 °C and

for 12 h at room temperature. The solvent was removed under re-

duced pressure, and the reaction crude was dissolved in DMF (3−4 mL).

Morpholine (143 mg, 1.65 mmol) and Et₃N (230 μL, 1.65 mmol) were

added, and the mixture was stirred for 12 h at room temperature. The

mixture was diluted with H₂O and EtOAc. The organic phase was

extracted four times with EtOAc (4 × 30 mL) and washed with H₂O (2 ×

30 mL) and brine (2 × 30 mL). The solvent was evaporated, and EtNH₂

(1.5 mL of a solution 2 M in MeOH) was added to the crude reaction

product. The mixture was heated for 24 h at 80 °C in a sealed tube.

The solvent and the excess of amine were removed under reduced

pressure. The residue was dissolved in EtOH (5 mL), and Pd(OH)₂/C

(0.01 mmol) was added. The mixture was stirred under an atmosphere

of hydrogen (balloon) for 1 h. The catalyst was ﬁltered oﬀ through

Celite, and the ethanol was removed under reduced pressure. The

reaction crude was puriﬁed by column chromatography (DCM/

MeOH, 90/10). The title compound was obtained as a purple oil

(174 mg, 68%). ¹H NMR (400 MHz, MeOD): δ 7.30−7.25 (m, 4 H),

6.82 (s, 1 H), 6.31 (s, 1 H), 4.84 (bs, 3 H), 3.63−3.61 (m, 4 H), 3.46

(s, 2 H), 3.37 (q, J = 7.2 Hz, 2 H), 3.10 (qn, J = 6.8 Hz, 1 H), 2.41−

2.39 (m, 4 H), 1.19 (t, J = 7.2 Hz, 3 H), 1.03 (d, J = 6.8 Hz, 6 H). ¹³C

NMR (100 MHz, MeOD): δ 161.1, 156.6, 150.8, 140.0, 137.8, 137.4,

129.4, 128.1, 126.3, 125.1, 125.0, 114.4, 101.8, 65.8, 62.0, 52.7, 33.2,

25.5, 21.1, 13.2. HRMS (ESI) calcd for C₂₅H₃₂N₅O₄(M + 1)⁺

466.2454; found 466.2452.

1

that solidiﬁed on standing (0.769 g, 76%). Mp 56−58 °C. H NMR

(400 MHz, CDCl₃): δ 7.51 (d, J = 8.0 Hz, 2 H), 7.30 (d, J = 8.0 Hz, 2

H), 4.72 (s, 2 H), 3.79 (s, 3 H), 0.91 (s, 9 H), 0.07 (s, 6 H). ¹³C NMR

(100 MHz, CDCl₃): δ 154.0, 144.2, 132.5, 125.5, 117.4, 86.3, 79.7,

63.9, 52.2, 25.4, 17.9, −5.8.

Methyl 1-[2,4-Bis(benzyloxy)cumenyl]-5-[p-(tert-butyl-

dimethylsilyloxymethyl)phenyl]-1H-1,2,3-triazole-4-carboxy-

late (15). Compound 14 (277 mg, 0.91 mmol) was added at room

temperature to a solution of azide 1 (373 mg, 1 mmol), dissolved in

DMF (2.5 mL). The ﬂask was subjected to three vacuum−nitrogen

cycles. Then [Cp*RuCl]₄(49 mg, 0.045 mmol) was added and the

reaction mixture was purged again three times. The reaction was left at

room temperature until completion (12 h, monitored by TLC).

EtOAc (20 mL) and water (10 mL) were then added. The organic

phase was extracted four times with EtOAc, washed with water (three

times) and brine (one time), and dried over Na₂SO₄. The solvent was

removed, and the mixture was puriﬁed by column chromatography

(PE/EtOAc, 60/40). The title compound was obtained as a purple oil

(419 mg, 68%). ¹H NMR (400 MHz, CDCl₃): δ 7.31−7.16 (m, 13 H),

6.94 (d, J = 7.2 Hz, 2 H), 6.41 (s, 1 H), 4.92 (s, 2 H), 4.72 (s, 4 H),

3.82 (s, 3 H), 3.30−3.23 (m, 1 H), 1.13 (d, J = 6.8 Hz, 6 H), 0.93 (s, 9

H), 0.09 (s, 6 H). ¹³C NMR (100 MHz, CDCl₃): δ 161.3, 157.47

151.2, 142.6, 136.0, 135.7, 135.1, 129.8, 129.4, 128.12 128.1, 127.6,

127.5, 126.7, 126.4, 125.6, 124.9, 124.1, 117.1, 98.5, 70.2, 69.9, 64.1,

51.4, 25.9, 25.5, 22.1, 17.97, −5.66. HRMS (ESI) calcd for

C₄₀H₄₇N₃O₅SiNa (M + Na)⁺701.3217 (43.3%), 700.3183 (100%);

found 701.3215 (43%), 700.3181 (100%).

Methyl 1-[2,4-Bis(benzyloxy)cumenyl]-5-[p-(hydroxymethyl)-

phenyl]-1H-1,2,3-triazole-4-carboxylate (16). TBAF (270 mg, 1.25

mmol) was added to a solution of 15 (460 mg, 0.68 mmol) in THF

(4 mL), and the mixture was stirred for 2 h at room temperature.

EtOAc (20 mL) was added, and the organic phase was washed with a

saturated solution of NH₄Cl and with H₂O. After the mixture was

dried over Na₂SO₄, the solvent was removed under reduced pressure

and the reaction crude was puriﬁed by column chromatography

(PE/EtOAc, 40:60). The title compound was obtained as a purple oil

(310 mg, 80%). ¹H NMR (400 MHz, CDCl₃): δ 7.29−7.15 (m, 13 H),

6.93 (d, J = 6.8 Hz, 2 H), 6.37 (s, 1 H), 4.89 (s, 2 H), 4.70 (s, 2 H),

4.62 (s, 2 H), 3.79 (s, 3 H), 3.31−3.18 (m, 1 H), 1.12 (d, J = 6.8 Hz,

6 H). ¹³C NMR (100 MHz, CDCl₃): δ 161.35, 157.57, 151.25, 142.69,

142.59, 136.01, 135.64, 135.10, 129.92, 129.53, 128.27, 128.16, 127.70,

Compound 19. Column chromatography with (DCM/MeOH,

1

90/10) gave compound 19 as a purple oil (166 mg, 63%). H NMR

(400 MHz, MeOD): δ 7.29 (AB system, J = 8.0 Hz, 4 H), 6.85 (s, 1

H), 6.33 (s, 1 H), 4.85 (bs, 3 H), 3.66−3.61 (m, 1 H), 3.51 (s, 2 H),

3.39 (q, J = 7.2 Hz, 2H), 3.11 (qn, J = 6.8 Hz, 1 H), 2.87−2.84 (m,

1 H), 2.64−2.61 (m, 1 H), 2.01−1.85 (m, 4H), 1.70−1.67 (m, 1 H),

1.54−1.44 (m, 1 H), 1.22 (t, J = 7.2 Hz, 3 H), 1.04 (d, J = 6.8 Hz, 6

H). ¹³C NMR (400 MHz, MeOD): δ 161.1, 156.5, 150.8, 140.8, 137.9,

137.4, 129.3, 128.2, 126.3, 125.0, 114.4, 101.9, 65.9, 61.7, 59.8, 52.3,

33.2, 31.9, 25.5, 22.0, 21.1, 13.2. HRMS (ESI) calcd for C₂₆H₃₄N₅O₄

(M + 1)⁺80.2611; found 480.2608.

Compound S-19. The product was isolated using (S)-3-

hydroxypiperidine and following the same procedure as 19 (161 mg,

61%). It showed the same spectroscopic features as 19. [a]²⁵_D+12.2 (c

0.1, MeOD).

Compound R-19. The product was isolated using (R)-3-

hydroxypiperidine and following the same procedure as 19 (146 mg,

55%). It showed the same spectroscopic features as 19. [a]²⁵−11.9

D

(c 0.1, MeOD).

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Compound 20. Column chromatography with (DCM/MeOH,

90/10) gave compound 20 as a purple oil (165 mg, 65%). H NMR

HRMS (ESI) calcd for C₂₅H₃₃N₆O₃(M + 1)⁺465.2614; found

465.2610.

1

(400 MHz, MeOD): δ 7.31 (AB system, J = 8.6 Hz, 4 H), 6.84 (s,

1 H), 6.33 (s, 1 H), 4.87 (bs, 3 H), 3.59 (s, 2 H), 3.39 (q, J = 7.2 Hz,

2 H), 3.10 (qn, J = 6.8 Hz, 1 H), 2.53−2.49 (m, 4 H), 1.61−1.57 (m,

4 H), 1.49−1.45 (m, 2 H), 1.21 (t, J = 7.2 Hz, 3 H), 1.06 (d, J = 6.8

Hz, 6 H). ¹³C NMR (100 MHz, MeOD): δ 161.0, 156.6, 150.9, 139.9,

137.4, 136.3, 129.4, 128.7, 126.3, 125.6, 125.0, 114.3, 101.9, 61.9, 53.2,

33.2, 25.5, 24.1, 22.8, 21.1, 13.21. HRMS (ESI) calcd for C₂₆H₃₄N₅O₃

(M + 1)⁺464.2662; found 464.2659.

Compound 21. Column chromatography with (DCM/MeOH,

97/3) gave compound 21 as a solid (212 mg, 85%). Mp 106−108 °C.

¹H NMR (400 MHz, MeOD): δ 8.32 (bs, 2H), 7.31 (AB system, J =

8.0 Hz, 4 H), 6.85 (s, 1 H), 6.32 (s, 1H), 3.66 (s, 2 H), 3.39 (q, J = 7.2

Hz, 2 H), 3.13−3.06 (m, 1 H), 2.58−2.54 (m, 4 H), 1.81−1.77 (m, 4

H), 1.21 (t, J = 7.2 Hz, 3 H), 1.06 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100

MHz, MeOD): δ 161.0, 156.6, 150.9, 140.0, 138.2, 137.4, 129.5, 128.0,

126.2, 125.2, 125.0, 114.4, 101.9, 59.0, 53.4, 33.2, 25.5, 22.1, 21.1, 13.1.

HRMS (ESI) calcd for C₂₅H₃₂N₅O₃(M + 1)⁺450.2505; found

450.2503.

Compound 27. Column chromatography (DCM/MeOH, 88/12)

gave compound 27 as a pale yellow solid (121 mg, 46%). H NMR

1

(400 MHz, MeOD): δ 8.10 (βσ, 2H), 7.29 (AB system, J = 8.0 Hz, 4

H), 6.84 (s, 1 H), 6.33 (s, 1 H); 3.50 (s, 2 H), 3.39 (q, J = 7.2 Hz, 2

H), 3.14−3.07 (m, 1 H), 2.60−2.40 (m, 9 H), 2.32 (s, 3 H), 1.21 (t,

J = 7.2 Hz, 3 H), 1.06 (d, J = 6.4 Hz, 6 H). ¹³C NMR (100 MHz,

MeOD): δ 161.0, 156.5, 150.8, 140.0, 138.0, 137.4, 129.4, 128.0, 126.3,

125.1, 124.9, 114.4, 101.91, 61.4, 53.6, 51.3, 43.8, 33.2, 25.5, 21.1, 13.2.

HRMS (ESI) calcd for C₂₆H₃₅N₆O₃(M + 1)⁺479.2771; found

479.2767.

Compound 28. Column chromatography (DCM/MeOH, 93/7)

gave compound 28 as a purple oil (58%). ¹H NMR (400 MHz,

CDCl₃): δ 7.37−7.15 (m, 6 H), 7.09 (s, 1 H), 6.41 (s, 1 H), 4.94 (s,

2 H), 4.75 (s, 2 H), 3.89 (s, 3 H), 3.28−3.21 (bm, 2H), 2.88 (d, J =

11.2 Hz, 2 H), 2.29 (s, 6 H), 2.22−2.16 (m, 1 H), 1.95 (t, J = 11.2 Hz,

2 H), 1.78 (d, J = 12.0 Hz, 2 H), 1.57−1.48 (m, 2 H), 1.11 (d, J = 6.8

Hz, 6 H). ¹³C NMR (100 MHz, CDCl₃): δ 161.3, 157.3, 151.3, 142.4,

139.8, 135.9, 135.64, 134.9, 130.0, 129.3, 128.2, 128.1, 127.9, 127.6,

127.5, 126.6, 126.4, 125.6, 124.0, 117.3, 98.5, 70.4, 69.9, 62.1, 61.9,

52.6, 51.5, 41.0, 27.6, 25.9, 22.1. HRMS (ESI) calcd for C₂₈H₃₉N₆O₃

(M + 1)⁺507.3084; found 507.3080.

Compound 22. Column chromatography on silica gel previously

conditioned with ammonia (DCM/MeOH, 95/5 to 90/10) gave

1

compound 22 as a white solid. Mp 110−112 °C (132 mg, 54%). H

Compound 29. Column chromatography on silica gel previously

conditioned with ammonia (DCM/MeOH, 85/15 to 60/40) gave

compound 29 as a waxy material that solidiﬁed on standing (113 mg,

42%). ¹H NMR (400 MHz, MeOD): δ 8.94 (bs, 2H), 7.29 (AB

system, J = 8.0 Hz, 4H), 6.84 (s, 1 H), 6.33 (s, 1 H), 3.59 (q, J = 7.2

Hz, 2 H), 3.39 (q, J = 7.2 Hz, 2 H), 3.13−3.07 (m, 1 H), 2.94−2.78

(m, 2 H), 2.71−2.65 (m, 1 H), 2.56−2.50 (m, 1 H), 2.36−2.32 (m, 1

H), 2.26 (s, 6 H), 2.09−1.97 (m, 1 H), 1.78−1.67 (m, 1 H), 1.21

(t, J = 7.2 Hz, 3 H), 1.07 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100 MHz,

CDCl₃): δ 161.0, 156.5, 150.8, 140.0, 138.8, 129.4, 127.6, 126.3, 125.0,

114.4, 101.9, 64.6, 59.0, 56.0, 52.2, 41.7, 33.2, 27.4, 25.5, 21.1, 13.1.

HRMS (ESI) calcd for C₂₇H₃₇N₆O₃(M + 1)⁺493.2923; found 493.2919.

Compound 30. Column chromatography (DCM/MeOH, 95/5)

gave compound 30 as a purple oil (79 mg, 30%). ¹H NMR (400 MHz,

MeOD): δ 7.98 (bs, 2H), 7.39 (AB system, J = 8.4 Hz, 4 H), 6.91 (s, 1

H), 6.30 (s, 1 H), 4.01 (s, 2 H), 3.39 (q, J = 7.2 Hz, 2 H), 3.14−3.05

(m, 1 H), 2.88−2.80 (m, 1 H), 2.08−2.03 (m, 3 H), 1.85−1.80 (m, 3

H), 1.68 (d, J = 12.0 Hz, 1 H), 1.35−1.26 (m, 5 H), 1.21 (t, J = 7.2 Hz,

3 H), 1.09 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100 MHz, MeOD): δ

160.9, 156.7, 150.9, 139.8, 137.6, 135.1, 130.0, 127.9, 126,4, 126.2,

125.1, 114.3, 101.8, 56.2, 33.2, 29.69, 25.6, 24.6, 23.9, 21.1, 13.1.

HRMS (ESI) calcd for C₂₇H₃₆N₅O₃(M + 1)⁺478.2818; found

478.2821.

Compound 31. Column chromatography (DCM/MeOH, 90/10)

gave compound 31 as a white solid (113 mg, 42%). Mp 77−79 °C. ¹H

NMR (400 MHz, MeOD): δ8.25 (bs, 2 H), 7.35 (AB system, J = 8.4

Hz, 4 H), 6.88 (s, 1 H), 6.31 (s, 1 H), 3.86 (s, 2 H), 3.39 (q, J = 7.2

Hz, 2 H), 3.15−3.05 (m, 1 H), 2.52 (d, J = 6.8 Hz, 2 H), 1.78−1.50

(m, 8H), 1.27−1.24 (m, 3 H), 1.21 (t, J = 7.2 Hz, 3 H), 1.08 (d, J = 6.8

Hz, 6 H), 0.99−0.87 (m, 2 H). ¹³C NMR (100 MHz, MeOD): δ

161.0, 156.6, 150.9, 139.9, 137.5, 137.1, 129.7, 127.6, 126.2, 125.7,

125.4, 114.3, 101.9, 54.0, 51.6, 35.9, 33.2, 30.2, 25.5, 25.0, 21.1,

13.1. HRMS (ESI) calcd for C₂₈H₃₈N₅O₃(M + 1)⁺492.2975; found

492.2977.

NMR (400 MHz, MeOD): δ 8.26 (bs, 2H), 7.71 (s, 1 H), 7.25 (AX

system, J = 8.0 Hz, 4 H), 7.06 (s, 1 H), 6.96 (s, 1 H), 6.88 (s, 1 H),

6.32 (s, 1 H), 5.17 (s, 2 H), 3.37 (q, J = 7.2 Hz, 2 H), 3.14−3.05 (m,

1 H), 1.20 (t, J = 7.2 Hz, 3 H), 1.06 (d, J = 6.8 Hz, 6 H). ¹³C NMR

(100 MHz, MeOD): δ 161.0, 156.6, 150.6, 139.7, 137.5, 136.7, 129.9,

127.3, 126.4, 126.1, 125.9, 125.0, 119.2, 114.3, 101.9, 49.4, 33.2, 25.5,

21.1, 13.2. HRMS (ESI) calcd for C₂₄H₂₇N₆O₃(M + 1)⁺447.2145;

found 447.2143.

Compound 23. Column chromatography (DCM/MeOH, 88/12)

was used. The title compound was obtained as a purple oil (162 mg,

1

58%). H NMR (400 MHz, MeOD): δ 7.36 (s, 5 H), 6.85 (s, 1 H),

6.33 (s, 1 H), 4.83 (bs, 4 H), 4.17 (d, J = 13.2 Hz, 1 H), 3.72−3.59 (m,

2 H), 3.56 (J = 13.2 Hz, 1 H), 3.39 (q, J = 7.2 Hz, 2 H), 3.10 (qn, J =

6.8 Hz, 1 H), 2.87−2.84 (m, 1 H), 2.76−2.72 (m, 1 H), 2.35−2.31

(m,1 H), 2.08−2.03 (m, 1 H), 1.82−1.35 (m, 6 H), 1.20 (t, J = 7.2 Hz,

3 H), 1.03 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100 MHz, MeOD): δ

160.9, 150.2, 149.9, 144.0, 138.1, 130.2, 130.0, 127.7, 127.4, 126.1,

122.0, 119.8, 105.7, 61.6, 60.2, 58.5, 52.0, 34.3, 33.3, 28.4, 25.4, 25.3,

22.8, 22.7, 14.7. HRMS (ESI) calcd for C₂₈H₃₈N₅O₄(M + 1)⁺

508.2924; found 508.2921.

Compound 24. Column chromatography (DCM/MeOH, 90/10)

1

gave compound 24 as a reddish oil (136 mg, 55%). H NMR (400

MHz, MeOD): δ 7.33 (AB system, J = 8.4 Hz, 4 H), 6.84 (s, 1 H),

6.32 (s, 1 H), 4.83 (bs, 3 H), 3.73 (s, 2 H), 3.39 (q, J = 7.2 Hz, 2 H),

3.10 (qn, J = 6.8 Hz, 1 H), 2.65 (q, J = 7.2 Hz, 4 H), 1.21 (t, J = 7.2

Hz, 3 H), 1.11−1.06 (m, 12 H). ¹³C NMR (100 MHz, MeOD): δ

160.9, 150.2, 149.9, 144.0, 143.3, 130.0, 128.6, 128.1, 127.0, 126.0,

122.0, 119.8, 105.7, 61.2, 46.7, 34.3, 25.3, 22.7, 14.7, 11.8. HRMS

(ESI) calcd for C₂₅H₃₄N₅O₃(M + 1)⁺452.2662; found 452.2664.

Compound 25. Column chromatography (DCM/MeOH, 90/10)

1

gave compound 25 as a purple oil (185 mg, 72%). H NMR (400

MHz, MeOD): δ 7.30 (s, 5 H), 6.85 (s, 1 H), 6.33 (s, 1 H), 4.88 (bs, 4

H), 3.63 (s, 2 H), 3.60 (t, J = 6.2 Hz, 2 H), 3.39 (q, J = 7.2 Hz, 2 H),

3.11 (qn, J = 6.8 Hz, 1 H), 2.61 (t, J = 6.2 Hz, 2 H), 2.54 (q, J = 7.2

Hz, 2 H), 1.21 (t, J = 7.2 Hz, 2 H), 1.07 (d, J = 6.8 Hz, 6 H), 1.02 (t, J

= 7.2 Hz, 3 H). ¹³C NMR (100 MHz, MeOD): δ 161.1, 156.5, 150.87,

140.1, 139.6, 137.4, 129.3, 127.8, 126.3, 125.0, 124.7, 114.4, 101.9,

58.6, 57.2, 54.2, 47.0, 33.2, 25.5, 21.1, 13.2, 9.9. HRMS (ESI) calcd for

C₂₅H₃₄N₅O₄(M + 1)⁺468.2611; found 468.2608

Compound 26. Column chromatography (DCM/MeOH, 75/25)

gave compound 26 as a white solid (102 mg 40%). Mp 79−81 °C. ¹H

NMR (400 MHz, MeOD): δ 8.16 (bs, 2H), 7.30 (AB system, J = 8.0

Hz, 4 H), 6.84 (s, 1 H), 6.35 (s, 1 H), 3.53 (s, 2 H), 3.39 (q, J = 7.2

Hz, 2 H), 3.17−3.07 (m, 1 H), 3.04 (t, J = 4.8 Hz, 4 H), 2.55 (m,6H),

1.21 (t, J = 7.2 Hz, 3 H), 1.06 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100

MHz, MeOD): δ 161.0, 156.6, 150.9, 140.0, 137.9, 137.4, 129.4, 127.9,

126.2, 125.1, 124.9, 114.4, 101.9, 61.4, 50.3, 43.4, 33.2, 25.5, 21.1, 13.1.

Compound 32. Column chromatography (DCM/MeOH, 80/20)

1

gave compound 32 as a white solid (120 mg, 43%). Mp < 50 °C. H

NMR (400 MHz, MeOD): δ 7.86 (bs, 2 H), 7.33 (AB system, J = 8.0

Hz, 4 H), 6.89 (s, 1 H), 6.31 (s, 1 H), 3.84 (s, 2 H), 3.65 (t, J = 4.4 Hz,

4 H), 3.39 (q, J = 7.2 Hz, 2 H), 3.14−3.07 (m, 2 H), 2.72 (m, 3 H),

2.48 (t, J = 6.4 Hz, 2 H), 2.38 (t, J = 4.4 Hz, 4 H), 1.21 (t, J = 7.2 Hz, 3

H), 1.08 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100 MHz, MeOD): δ 161.0,

156.6, 150.8, 140.0, 138.4, 137.6, 129.7, 127.3, 126.3, 125.4, 125.0,

114.3, 101.8, 65.9, 55.9, 52.8, 51.5, 43.4, 33.2, 25.5, 21.1, 13.1. HRMS

(ESI) calcd for C₂₇H₃₇N₆O₄(M + 1)⁺509.2876; found 509.2876.

Compound 33. Column chromatography on silica gel previously

conditioned with ammonia (DCM/MeOH, 90/10 to 80/20) gave

compound 33 as a white solid (87 mg, 32%). Mp 69−71 °C. ¹H NMR

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(400 MHz, MeOD): δ 8.1 (bs, 2 H), 7.3 (AB system, J = 8.0 Hz, 4 H),

6.8 (s, 1 H), 6.2 (s, 1 H), 3.7 (s, 2 H), 3.3 (q, J = 2 H), 3.1−3.0 (m, 1

H), 2.6−2.5 (m, 6 H), 2.5 (q, J = 7.2 Hz, 4 H), 1.2 (t, J = 7.2 Hz, 3 H),

1.0 (d, J = 7.2 Hz, 6 H), 1.0 (t, J = 7.2 Hz, 6 H). ¹³C NMR (100 MHz,

MeOD): δ 161.1, 156.7, 142.0, 140.0, 137.5, 129.5, 127.0, 125.7, 124.9,

114.5, 102.2, 52.2, 50.9, 46.2, 44.8, 33.2, 25.5, 21.2, 13.1, 9.6. HRMS

(ESI) calcd for C₂₇H₃₉N₆O₃(M + 1)⁺495.3084; found 495.3086.

Compound 34. Column chromatography on silica gel previously

conditioned with ammonia (DCM/MeOH, 90:10 to 80:20) gave

compound 34 as a white solid (89 mg, 35%). Mp 56−58 °C. ¹H NMR

(400 MHz, MeOD): δ 7.95 (bs, 2 H), 7.31 (AB system, J = 8.0 Hz,

4 H), 6.74 (s, 1 H), 6.25 (s, 1 H), 3.71 (s, 2 H), 3.38 (q, J = 7.2 Hz,

2 H), 3.11−3.04 (m, 1 H), 2.57−2.45 (m, 10 H), 1.69−1.62 (m, 2 H),

1.21 (t, J = 7.2 Hz, 3 H), 1.06−1.00 (m, 12 H). ¹³C NMR (100 MHz,

MeOD): δ 161.2, 156.8, 140.0, 139.9, 129.6, 127.0, 124.8, 124.7, 114.8,

102.9, 52.7, 52.2, 50.1, 45.8, 33.2, 25.4, 24.8, 21.2, 13.1, 9.4. HRMS

(ESI) calcd for C₂₈H₄₁N₆O₃(M + 1)⁺509.3240; found 509.3238.

Compound 35. Column chromatography on silica gel previously

conditioned with ammonia (DCM/MeOH, 50/50) gave compound

35 as a white solid (95 mg, 35%). Mp 67−69 °C. ¹H NMR (400 MHz,

MeOD): δ 8.08 (bs, 2 H), 7.31 (AB system, J = 8.4 Hz, 4 H), 6.87 (s,

1 H), 6.30 (s, 1 H), 3.76 (s, 2 H), 3.39 (q, J = 7.2 Hz, 2 H), 3.16−3.07

(m, 1 H), 2.86−2.83 (m, 2 H), 2.49−2.42 (m, 2 H), 2.25 (s, 3 H), 2.01

(t, J = 11.2 Hz, 2 H), 1.91−1.87 (m, 2 H), 1.48−1.38 (m, 3 H), 1.21

(t, J = 7.2 Hz, 3 H), 1.08 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100 MHz,

MeOD): δ 156.6, 150.8, 140.0, 137.8, 137.63, 129.7, 127.4, 126.3,

125.6, 125.1, 114.3, 101.8, 52.7, 52.1, 48.6, 43.3, 33.2, 28.8, 25.5, 21.1,

13.1. HRMS (ESI) calcd for C₂₇H₃₇N₆O₃(M + 1)⁺493.2927; found

493.2923.

Methyl 1-[2,4-Bis(benzyloxy)cumenyl]-5-[p-(morpholin-4-

ylmethyl)phenyl]-1H-1,2,3-triazole-4-carboxylate (36). Et₃N

(230 μL, 1.65 mmol) and MsCl (130 μL, 1.65 mmol) were added

to a solution of 16 (310 mg, 0.55 mmol) in DCM (7 mL) at 0 °C. The

solution was stirred for 30 min at 0 °C and for 12 h at room

temperature. The solvent was removed under reduced pressure, and

the reaction crude was dissolved in DMF (3−4 mL). Morpholine

(143 mg, 1.65 mmol) and Et₃N (230 μL, 1.65 mmol) were added, and

the mixture was stirred for 12 h at room temperature. The mixture was

diluted with H₂O and EtOAc. The organic phase was extracted four

times with EtOAc (4 × 30 mL) and washed with H₂O (2 × 30 mL)

and with brine (2 × 30 mL). The solvent was evaporated. Column

chromatography (DCM/MeOH, 98/2) gave the title compound as a

pale yellow oil (372 mg, 75%). ¹H NMR (400 MHz, CD₃Cl): δ 7.37−

7.22 (m, 10 H), 7.17 (d, J = 8.4 Hz, 2 H), 7.08 (s, 1 H), 7.01−6.99 (m,

2 H), 6.40 (s, 1 H), 4.93 (s, 2 H), 4.74 (s, 2 H), 3.88 (s, 3 H), 3.67 (t,

J = 4.4 Hz, 4 H), 3.47 (s, 2 H), 3.24 (qn, J = 6.8 Hz, 1 H), 2.42−2.38

(m, 4 H), 1.10 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100 MHz, CD₃Cl): δ

160.6, 158.4, 156.9, 151.3, 140.0, 139.2, 136.6, 136.0, 131.9, 130.7,

128.4, 128.4, 128.4, 128.0, 127.8, 127.8, 127.6, 126.6, 125.2, 121.1,

100.2, 70.3, 70.1, 67.1, 63.2, 53.8, 52.0, 26.3, 22.8.

chromatography (DCM/MeOH, 96/4) to give compound 37 as a

waxy material (11 mg, 31% yield). ¹H NMR (300 MHz, DMSO-d₆): δ

9.70 (bs, 2H), 8.75 (t, J = 5.8 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.20

(d, J = 8.4 Hz, 2H), 6.90 (s, 1H), 6.37 (s, 1H), 3.70 (t, J = 6.0, J = 6.7

Hz, 2H), 3.51−3.57 (m, 6H), 3.4 (bs, 2H), 3.00 (hept, J = 6.9 Hz,

1H), 2.27−2.30 (m, 4H), 0.99 (d, J = 6.9 Hz, 6H). ¹³C NMR (75

MHz, DMSO-d₆): δ 160.7, 150.2, 149.9, 142.0, 140.3, 130.3, 130.0,

128.4, 127.8, 127.7, 122.0, 120.6, 105.7, 67.1, 63.2, 53.8, 43.7, 41.9,

25.3, 22.7. HRMS (ESI) calcd for C₂₅H₃₁ClN₅O₄(M + 1)⁺502.2035

(32.4%), 500.2065 (100%); found 502.2034 (32%), 500.2061 (100%).

Compound 38. Column chromatography (DCM/MeOH, 98/2)

1

gave compound 38 as a waxy material (15 mg, 42%). H NMR (300

MHz, DMSO-d₆): δ 9.78 (bs, 1H), 9.70 (bs, 1H), 8.49 (t, J = 5.9 Hz,

1H), 7.23 (d, J = 8.5 Hz, 2H), 7.19 (d, J = 8.5 Hz, 2H), 6.87 (s,

1H),6.37 (s, 1H), 3.51−3.54 (m, 4H), 3.39 (s, 2H), 3.16−3.22 (m,

2H), 3.00 (hept, J = 6.7 Hz, 1H), 2.29−2.30 (m, 4H), 1.45−1.48 (m,

2H), 1.22−1.25 (m, 7H), 1.01 (d, J = 6.9 Hz, 6H), 0.84 (t, J = 6.6 Hz,

3H). ¹³C NMR (75 MHz, DMSO-d₆): δ 160.7, 150.2, 149.9, 143.9,

140.3, 130.4, 130.0, 128.4, 127.7, 127.7, 122.0, 120.6, 105.7, 67.1, 63.2,

53.8, 40.7, 31.7, 27.6, 26.0, 25.3, 22.8, 22.7, 14.1. HRMS (ESI) calcd

for C₃₀H₄₁N₅O₄Na (M + Na)⁺559.3090 (32.4%), 558.30565 (100%);

found 559.3087 (33%), 558.30567 (100%).

Compound 39. Column chromatography (DCM/MeOH, 96/4)

1

gave compound 39 as a waxy material (13 mg, 38%). H NMR (300

MHz, DMSO-d₆): δ 9.69 (s, 1H),9.66 (s, 1H), 8.28 (d, J = 7.8 Hz,

1H), 7.23 (d, J = 8.5 Hz, 2H), 7.20 (d, J = 8.5 Hz, 2H), 6.87 (s, 1H),

6.37 (s, 1H), 4.10−4.22 (m, 1H), 3.52−3.55 (m, 4H), 3.41 (bs, 2H),

3.00 (hept, J = 6.9 Hz, 1H), 2.30 (bs, 4H), 1.45−1.90 (m, 8H), 0.99

(d, J = 6.9 Hz, 6H). ¹³C NMR (75 MHz, DMSO-d₆): δ 160.8, 150.2,

149.9, 144.6, 140.3, 130.4, 130.0, 128.4, 127.8, 127.7, 122.1, 120.6,

105.7, 67.1, 63.2, 53.8, 50.2, 32.6, 25.3, 23.1, 22.7. HRMS (ESI) calcd

for C₂₈H₃₅N₅O₄Na (M + Na)⁺529.2620 (30.3%), 528.2587 (100%);

found 529.2623 (31%), 528.2590 (100%).

Compound 40. Column chromatography (DCM/MeOH, 98/2)

1

gave compound 40 as a waxy material (15 mg, 42%). H NMR (300

MHz, DMSO-d₆): δ 9.74 (bs, 1H), 9.69 (bs, 1H), 8.19 (d, J = 8.6 Hz,

1H), 7.23 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.4 Hz, 2H), 6.87 (s, 1H),

6.37 (s, 1H), 3.69 (bs, 1H), 3.51−3.54 (m, 4H), 3.39 (bs, 2H), 3.00

(hept, J = 6.7 Hz, 1H), 2.28−2.30 (m, 4H), 1.54−1.75 (m, 5H), 1.18−

1.41 (m, 5H), 0.99 (d, J = 6.7 Hz, 6H). ¹³C NMR (75 MHz, DMSO-

d₆): δ 160.8, 150.2, 149.9, 144.6, 140.3, 130.4, 130.0, 128.4, 127.8,

127.7, 122.1, 120.6, 105.7, 67.1, 63.2, 53.8, 48.1, 32.4, 25.4, 25.3, 25.2,

22.7. HRMS (ESI) calcd for C₂₉H₃₈N₅O₄(M + 1)⁺520.2924; found

520.2927.

Compound 41. Column chromatography (DCM/MeOH, 95/5)

1

gave compound 41 as a waxy material (8 mg, 22%). H NMR (300

MHz, DMSO-d₆): δ 9.76 (bs, 2H), 7.26 (d, J = 8.1 Hz, 2H), 7.17 (d,

J = 8.1 Hz, 2H), 6.99 (s, 1H), 6.40 (s, 1H), 3.51−3.54 (m, 8H), 3.41

(bs, 2H), 3.2−3.3 (bm, 4H), 3.04 (hept, J = 6.9 Hz, 1H), 2.27−2.30

(m, 4H), 1.03 (d, J = 6.9 Hz, 6H). ¹³C NMR (75 MHz, DMSO-d₆): δ

162.6, 150.2, 149.9, 143.5, 140.3, 130.3, 130.0, 128.4, 127.8, 127.7,

122.0, 120.6, 105.7, 67.1, 66.4, 63.2, 53.8, 45.3, 44.6, 25.3, 22.8. HRMS

(ESI) calcd for C₂₇H₃₄N₅O₅(M + 1)⁺508.2560; found 508.2563.

Compound 42. The desired amide was obtained after puriﬁcation

by preparative HPLC using a gradient of a binary mixture of H₂O/

CH₃CN further containing 0.1% TFA from 90/10 to 10/90. The yield

(2-Chloroethylamino)(1-(2,4-dihydroxycumenyl)-5-[p-(mor-

pholin-4-ylmethyl)phenyl]-1H-1,2,3-triazol-4-yl)formaldehyde

(37). General Procedure. Compound 36 (50 mg, 0.07 mmol) was

dissolved in a mixture of THF/H₂O (2 mL) containing LiOH (5 mg,

0.2 mmol) and stirred at room temperature for 12 h. The solvent was

evaporated under vacuum and the crude product treated directly with

oxalyl chloride (1 mL). The vial was stirred for 2 h and the oxalyl

chloride evaporated under vacuum. The residue was dissolved in dry

DCM (2 mL), cooled to 0 °C, and DIPEA (0.2 mL, 2 mmol) and

2-chloroethylamine hydrochloride (58 mg, 0.5 mmol) were subsequently

added. The mixture was stirred at room temperature for 12 h. Then

the solvent was evaporated and the residue treated with EtOAc and

passed through a short cartridge containing silica gel (eluting with

EtOAc). The collected eluate was dried on anhydrous MgSO₄, and the

solvent was evaporated and treated several times with dry toluene that

was further evaporated in order to remove azeotropically any trace of

water. The residue was dissolved in DCM (0.1 mL), and the mixture

was cooled to 0 °C and treated with 0.2 mL of a 1 M solution of BCl₃

in DCM. After the mixture was stirred for 2 h at room temperature,

the solvent was evaporated and the residue puriﬁed by column

1

was 7 mg of 42 (19%). H NMR (300 MHz, MeOD): δ 7.44−7.51

(m, 4H), 7.46 (d, J = 8.7 Hz, 2H), 6.97 (s, 1H), 6.31 (s, 1H), 4.49 (m,

1H), 4.33 (bs, 2H), 3.88 (bs, 4H) 3.27 (bs, 4H), 3.12 (hept, J = 6.9

Hz, 1H), 2.31 (m, 1H), 1.10 (d, J = 6.9 Hz, 6H), 1.04 (d, J = 6.9 Hz,

6H). ¹³C NMR (75 MHz, DMSO-d₆): δ 174.5, 158.9, 150.2, 149.9,

142.9, 140.3, 130.3, 130.0, 128.4, 127.8, 127.7, 122.0, 120.6, 105.7,

67.1, 63.2, 58.6, 53.8, 31.2, 25.3, 22.8, 19.3, 18.2. HRMS (ESI) calcd

for C₂₈H₃₅N₅O₆M⁺537.2588; found 537.2586.

Compound 45. Methyl 7-aminoheptanoate HCl (31 mg, 0.16

mmol), DIPEA (51 μL, 0.28 mmol), and PyBOP (62 mg, 0.12 mmol)

were added to a solution of acid obtained from 36 as previously

described (62 mg, 0.10 mmol) in 4 mL of DCM. The reaction mixture

was stirred at room temperature until complete conversion (6 h, TLC

check with eluent DCM/MeOH, 96/4) and then diluted with DCM

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and washed with H₂O. The organic phase was dried over Na₂SO₄and

concentrated at reduced pressure. The crude product was puriﬁed by

ﬂash chromatography (DCM/MeOH, 98/2) to give 45 (46 mg, 58%).

¹H NMR (300 MHz, DMSO-d₆): δ 8.57 (t, 1H, J = 5.7 Hz), 7.43 (m,

5H), 7.31 (m, 5H), 7.23 (s, 1H), 7.19 (m, 4H), 6.91 (s, 1H), 5.13 (s,

2H), 4.97 (s, 2H), 3.57 (s, 3H), 3.54 (m, 4H), 3.43 (s, 2H), 3.21 (m,

2H), 3.16 (m, 1H), 2.31 (bm, 4H), 2.28 (t, 2H, J = 7.3 Hz), 1.51 (m,

4H), 1.27 (m, 4H). 1.06 (d, 6H, J = 7.1 Hz). ¹³C NMR (75 MHz,

MeOD): δ 173.5, 160.7, 158.4, 156.8, 143.9, 140.3, 136.6, 136.0, 131.2,

130.8, 128.4, 128.3, 127.9, 127.8, 127.7, 127.6, 126.6, 124.9, 120.9,

100.4, 70.3, 70.1, 67.1, 63.2, 53.8, 51.1, 40.4, 33.6, 28.8, 27.0, 26.3,

25.4, 25.0, 22.8. ESI-MS m/z = 782.4 [M + Na]⁺.

Compound 46. BCl₃at 1 M in DCM (240 μL, 0.24 mmol) was

added to a solution of 45 (46 mg, 0.06 mmol) in DCM (2 mL), and

the mixture was stirred at 0 °C. The reaction mixture was allowed to

warm to room temperature, and after 3 h the conversion of 45 was

complete. Ethyl acetate was added, and the mixture was washed with

a saturated solution of NaHCO₃. The aqueous phase was extracted

3 times with EtOAc, and organic phases were collected, dried over

Na₂SO₄, ﬁltered, and concentrated at reduced pressure. The crude

product was puriﬁed by ﬂash chromatography (DCM/MeOH, 95/5)

to give the desired product (35 mg, 93%). ¹H NMR (300 MHz,

DMSO-d₆): δ 9.70 (bs, 1H), 9.65 (s, 1H), 8.51 (t, 1H, J = 5.8 Hz),

7.21 (m, 4H), 6.84 (s, 1H), 6.38 (s, 1H), 3.56 (s, 3H), 3.51 (bs, 4H),

3.40 (bs, 2H), 3.20 (m, 2H), 3.02 (m, 1H), 2.25 (m, 6H), 1.47 (m,

4H), 1.28 (m, 4H), 1.05 (d, 6H, J = 7.0 Hz). ESI-MS m/z = 782.4

[M + Na]⁺, 758.3 [M − H]⁻.

AUTHOR INFORMATION

■

Corresponding Authors

*M.T.: phone, +39-0577234275; fax, +39-0577234333; e-mail,

maurizio.taddei@unisi.it.

*G.G.: phone, +39-0691394441; fax, +39-0691393638; e-mail,

giuseppe.giannini@sigma-tau.it.

Present Addresses

^∥M.C.: Dipartimento di Chimica “Ugo Schiﬀ”, Universita

̀

degli

Studi di Firenze, Via della Lastruccia 3, Sesto Fiorentino (FI),

Italy.

^⊥C.P.: Biogem S.C.A.R.L., Via Camporeale, I-83031 Ariano

Irpino (AV), Italy.

^#W.C.: Indena, SpA, Viale Ortles 12, I-20139 Milano, Italy.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by grants from Sigma-Tau Research

Switzerland S.A. that retains the property rights of all the

compounds described in the paper. The authors thank Dr.

Tiziana Brunetti and Dr. Gianfranco Battistuzzi for their useful

support in the preparation of the manuscript.

REFERENCES

Compound 43. Hydroxylamine (50% acqueous solution, 50 μL,

0.75 mmol) and 1 M NaOH (500 μL, 0.50 mmol) were added to a

stirred solution of 46 (30 mg, 0.05 mmol) in MeOH (500 μL, 0.1 M)

at 0 °C. The reaction mixture was warmed to room temperature, and

after 2 h, TLC analysis showed complete conversion of starting

material. The solution was extracted with AcOEt, and the organic

phase was washed with water and brine, dried over Na₂SO₄, and

concentrated under reduced pressure. The crude product was puriﬁed

by preparative HPLC with TFA buﬀered eluents to give saliﬁed 43 (18

■

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regulation of apoptosis: new strategies in tumor therapy: a

comprehensive review. Pharmacol. Ther. 2004, 101, 227−257.

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mediated targeted disruption of multiple protein activities through

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hot. Clin. Cancer Res. 2009, 1, 9−14.

1

mg, 49%). H NMR (300 MHz, DMSO-d₆): δ 10.35 (bs, 2H), 9.75

(bs, 2H), 8.58 (t, J = 5.6 Hz, 1H), 7.40 (m, 4H), 6.94 (s, 1H), 6.39 (s,

1H), 4.30 (s, 2H), 3.92 (bm, 2H), 3.65 (bm, 2H), 3.17 (m, 2H), 3.10

(bm, 4H), 3.01 (m, 1H), 1.90 (t, J = 7.3 Hz, 2H), 1.43 (m, 4H), 1.24

(m, 4H). 1.09 (d, J = 6.9 Hz, 6H). ¹³C NMR (75 MHz, DMSO-d₆): δ

172.4, 160.7, 150.2, 149.9, 143.9, 140.3, 130.4, 130.0, 128.4, 127.8,

127.7, 122.1, 120.6, 105.7, 67.1, 63.3, 53.8, 40.4, 32.1, 27.2, 25.5, 25.4,

25.3, 24.5, 22.8. ESI-MS m/z = 581.3 [M + H]⁺. ESI-MS m/z = 579.4

[M − H]⁻. HRMS (ESI) calcd for C₃₀H₄₁N₆O₆(M + 1)⁺581.3088;

found 581.3085.

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Compound 44. Compound 18 (46 mg, 0.1 mmol) was dissolved

in acetic anhydride (1 mL) with a catalytic amount of perchlorate (0.2

mmol), and the mixture was stirred at room temperature for 12 h. The

acetic anhydride solution was treated with 1 N HCl in order to

transform it into acetic acid that was removed by co-distillation with

hexane (rotavapor). The solid resulting 44 (48 mg, 92%) was obtained

1

with 100% purity. H NMR (400 MHz, MeOD): δ 7.30−7.25 (m, 4

H), 6.82 (s, 1 H), 6.31 (s, 1 H), 4.84 (bs, 3 H), 3.63−3.61 (m, 4 H),

3.46 (s, 2 H), 3.37 (q, J = 7.2 Hz, 2 H), 2.41−2.39 (m, 4 H), 2.08 (s, 6

H), 1.19 (t, J = 7.2 Hz, 3 H), 1.03 (d, J = 6.8 Hz, 6 H). ¹³C NMR (100

MHz, MeOD): δ 161.0, 156.5, 150.8, 140.0, 137.8, 137.4, 129.4, 128.1,

126.3, 125.1, 125.0, 114.4, 101.8, 65.8, 62.0, 52.7, 33.2, 25.5, 21.1, 20.3,

13.2. HRMS (ESI) calcd for C₂₉H₃₆N₅O₆(M + 1)⁺550.2666; found

550.2665.

ASSOCIATED CONTENT

■

S

* Supporting Information

Synthetic scheme of compound 43, analysis of Hsp90 client

protein levels, and the antiproliferative activity of all 1,2,3-

triazoles on a panel of tumor cell lines. This material is available

free of charge via the Internet at http://pubs.acs.org.

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Journal of Medicinal Chemistry

Article

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curve ﬁtting using the GraphPad Prism software program suggested a

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dx.doi.org/10.1021/jm401536b | J. Med. Chem. 2014, 57, 2258−2274

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Synthesis and evaluation of new Hsp90 inhibitors based on a 1,4,5-trisubstituted 1,2,3-triazole scaffold

DOI: 10.1021/jm401536b

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